Naturally occurring adenines within mRNA coding sequences affect ...

JB Accepts, published online ahead of print on 3 November 2006 J. Bacteriol. doi:10.1128/JB.01356-06 Copyright © 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Naturally occurring adenines within mRNA coding sequences affect ribosome binding and expression in Escherichia coli

Authors: Jay E. Brock1, Robert L. Paz2, Patrick Cottle, and Gary R. Janssen*

Running Title: Adenine stimulation of gene expression

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Key Words: translation initiation, ribosome binding, gene expression, adenine

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stimulation, translation enhancer

Author Affiliation: 1Department of Microbiology Miami University 32 Pearson Hall Oxford, Ohio 45056 USA

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Phone: 513-529-5448 Fax: 513-529-2431 Email: [email protected]

Address for correspondence: Gary Janssen Department of Microbiology Miami University 32 Pearson Hall Oxford, Ohio 45056 USA Phone: 513-529-5422 Fax: 513-529-2431 Email: [email protected]

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The Fuqua School of Business Duke University Box 90120 Durham, NC 27708-0120

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*corresponding author

Abstract

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Translation initiation requires the precise positioning of a ribosome at the start codon. The major signals of bacterial mRNA that direct the ribosome to a translational

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start site are the Shine-Dalgarno (SD) sequence, within the untranslated leader, and the

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start codon. Evidence for many non-SD-led genes in prokaryotes provides motive for

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studying additional interactions between ribosomes and mRNA that contribute to

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translation initiation. A high incidence of adenines have been reported downstream of

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the start codon for many E. coli genes and addition of downstream adenine-rich

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sequences increases expression from several genes in E. coli. We describe here site-

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directed mutagenesis of the E. coli aroL, pncB and cysJ coding sequences to assess the

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contribution of naturally occurring adenines to in vivo expression and in vitro ribosome

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binding from mRNAs with different SD-containing untranslated leaders. Base

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substitutions that decreased the downstream adenines by one or two nucleotides

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decreased expression significantly from aroL-, pncB- and cysJ-lacZ fusions; mutations

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increasing downstream adenines by one or two nucleotides increased expression

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significantly from aroL- and cysJ-lacZ fusions. Using primer extension inhibition

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(toeprint) and filter binding assays to measure ribosome binding, the changes in in vivo

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expression correlated closely with changes in in vitro ribosome binding strength. Our

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data are consistent with a model in which downstream adenines influence expression

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through their effects on the mRNA-ribosome association rate and the amount of ternary

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complex formed. This work provides evidence that adenine-rich sequence motifs might

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serve as a general enhancer of E. coli translation.

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Introduction

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Initiation of bacterial protein synthesis requires the selection of an mRNA’s translation initiation region (TIR) and initiator tRNA (fMet-tRNAfMet) by the 30S

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ribosomal subunit aided by the three initiation factors (IF1, IF2 and IF3). Initiation is the

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rate-limiting step of translation, and the formation of ribosome/initiator tRNA/mRNA

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ternary complexes is influenced by sequence and structural motifs in and around the

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mRNA’s ribosome binding site (RBS) (33). Features of the mRNA RBS contributing to

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the efficiency of ribosome binding and translation include the start codon (i.e., AUG most

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frequently in Escherichia coli) and the purine-rich Shine-Dalgarno (SD) sequence,

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located within the untranslated leader region (UTR), which base-pairs with the anti-SD

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(ASD) sequence near the 3’ end of 16S rRNA (29, 11). In addition to the start codon and

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SD:ASD interaction, other sequence and structural motifs within mRNA have been

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suggested to influence ribosome binding and translation including mRNA secondary

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structure within the TIR (6, 7), specific translation-enhancing sequences upstream (10,

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21, 37, 12) and downstream (32, 9, 19, 26) to the start codon, upstream pyrimidine-rich

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tracts (1, 42, 38), AU-rich sequences within the UTR (14, 15), and downstream A (3) and

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CA-rich tracts (16). In addition, an increasing number of non-SD-led genes (2) and genes

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encoding mRNA lacking a 5’-untranslated leader region (leaderless mRNA) (13, 18) are

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being identified, raising the potential for novel sequence and structural motifs within the

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coding sequence that contribute to the formation of translation initiation complexes.

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Statistical analysis of E. coli translational start sites (24, 27) revealed that the

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region downstream of the initiation codon for several genes contain CA- and/or A-rich

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sequences. In a recent analysis of nucleotide sequences around the boundaries of all open

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reading frames in E. coli, nucleotide biases were observed immediately downstream of

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the initiation codon and the two most frequent second codons, AAA and AAU, were

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found to enhance translational efficiency (26). In addition to these studies, adenine-rich

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sequences have been proposed to enhance translation in E. coli, as demonstrated by the

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increased expression observed for the human gamma interferon (γ-IFN) and

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chloramphenicol acetyltransferase (cat) genes after the addition of A-rich motifs

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downstream of the initiation codon (3).

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The insertion of CA multimers immediately downstream of the lacZ initiation

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codon results in an increase in gene expression (16, 30; G. R. Janssen, unpublished data).

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CA nucleotides, inserted downstream of the start codon for leaderless or SD-leadered

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mRNAs, exerted a greater stimulation when close to the initiation codon and exhibited a

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dose-dependent increase in expression with an increasing number of CA repeats.

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Increased expression was observed also after the addition of CA repeats to SD-leadered

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and leaderless neo, kan and gusA genes indicating that CA-rich sequences might serve as

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a general enhancer of expression for leadered and leaderless mRNAs in E. coli (16).

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The work of Chen et al. (3) and Martin-Farmer and Janssen (16) demonstrate that

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the addition of adenines and CA repeats, respectively, can dramatically increase gene

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expression. The experiments described here investigate the influence of naturally

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occurring downstream adenines on in vivo expression and in vitro ribosome binding. The

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mRNAs encoded by the E. coli aroL, pncB and cysJ genes include SD-containing

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untranslated leaders and adenines downstream of their AUG start codons; site-directed

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mutagenesis was used to decrease or increase the A-richness in order to generate putative

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“down” or “up” mutations, respectively. The putative “down” mutations in aroL, pncB

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and cysJ mRNAs decreased both in vivo expression and in vitro ribosome binding,

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whereas putative “up” mutations in the aroL and cysJ mRNAs increased both in vivo

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expression and in vitro ribosome binding. Using toeprint, filter binding and expression

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assays, our data show that downstream adenines contribute significantly to the rate and

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amount of ternary complex formed as well as the in vivo expression levels for the aroL,

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pncB and cysJ genes even in the presence of a canonical SD sequence.

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Materials and Methods

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Bacterial strains. E. coli DH5α [F-, phi80d, lacZM15∆ (lacZYA-argF), U169, recA1,

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endA1, hsdR17 (rk-, mk+), supE44, lambda-, thi-1, gyrA, relA1] was used as the host

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strain for all plasmid constructs. E. coli RFS859 [F-, thr-1, araC859, leuB6, ∆lac74, tsx-

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274, lambda-, gyrA111, recA11, relA1, thi-1], a lac-deletion strain (28), was used as the

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host for the expression and assay of β-galactosidase activity from lacZ reporter genes. E.

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coli K12 total genomic DNA was used for the isolation of wild type (WT) aroL, cysJ and

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pncB gene fragments. E. coli MRE600 (39) was used for the isolation of 30S ribosomal

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subunits.

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Reagents and recombinant DNA procedures. Radiolabelled nucleotides, [γ-32P]ATP

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(6,000 Ci/mmol; 150 mCi/ml) and [α-32P]CTP (3,000 Ci/mmol; 10 mCi/ml), were

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purchased from Perkin Elmer. Oligonucleotides were synthesized using a Beckman

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1000M DNA synthesizer or purchased from commercial suppliers. Restriction

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endonucleases, T4 DNA ligase, T4 polynucleotide kinase, T4 DNA polymerase and T7

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RNA polymerase were obtained from New England Biolabs. Pfu DNA polymerase was

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obtained from Stratagene. AMV reverse transcriptase was obtained from Life Sciences

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and RNase-free DNase I was purchased from Ambion. All enzymes were used according

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to the manufacturer’s recommendations. Plasmid isolation, E. coli transformations and

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other DNA manipulations were carried out in a standard manner (25).

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Mutant Constructions. The aroL-, cysJ- and pncB-lacZ fusions were constructed

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containing either the lacZ untranslated leader or the gene’s natural untranslated leader

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and the first sixteen codons of the coding sequence fused to a lacZ reporter gene.

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Transcription of the lac fusions was provided by the E. coli lac promoter. Plasmids

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(pBR322-derived) containing the aroL-, cysJ- and pncB-lacZ fusions were used as

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templates for site-directed mutagenesis in which one oligonucleotide primer contained

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the desired mutation(s) (Table 1) and the other primer annealing within the lacZ coding

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sequence. After amplification, the PCR product was trimmed with appropriate restriction

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enzymes to facilitate cloning, ultimately producing identical plasmids varying only by the

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single or double nucleotide mutations made within the coding sequence as shown in

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Table 1. All DNA regions generated by PCR amplification were verified by DNA

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sequencing.

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β-galactosidase assays. Triplicate cultures of plasmid-containing strains were grown in

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2XYT (per liter, 16g of Difco Bacto Tryptone, 10 g of Difco Bacto yeast extract, 10 g of

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NaCl, pH 7.4) supplemented with 200 µg/ ml ampicillin and 0.2 mM IPTG, at 37°C to an

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O.D.600 of 0.4-0.6 and quick chilled on ice. Triplicate β-galactosidase assays (17) were

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performed with each of the triplicate cultures.

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Primer extension inhibition (toeprinting). The mRNAs used in primer extension

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inhibition (toeprint) experiments were generated in vitro with T7 RNA polymerase.

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DNA templates for T7 transcription were prepared by PCR amplification of miniprep

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plasmid DNA. Oligonucleotide primers T7lac.lead (5’-

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ggaattctaatacgactcactatagaattgtgagcggataacaatttc-3’) and lac.comp2 (5’-

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attaagttgggtaacgccag-3’) were used to generate DNA fragments containing the T7

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promoter, lac leader and cysJ sequences (with or without mutations) fused to lacZ from

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the respective template plasmids. T7pncB.lead (5’-

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ctaatacgactcactatagttcctgaagatgtttattgtac-3’) and lac.comp2 were used to generate DNA

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fragments containing the T7 promoter, pncB leader and pncB sequences (with or without

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mutations) fused to lacZ. T7aroL.lead (5’-

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ggaattctaatacgactcactatagattgagattttcactttaagtgg-3’) and lac.comp2 were used to generate

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DNA fragments containing the T7 promoter, aroL leader and aroL sequences (with or

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with out mutations) fused to lacZ. After gel purification of the resulting PCR-generated

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fragments, transcription reactions [15 mM DTT, 4 mM NTPs, 40 mM Tris-HCL (pH7.9),

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20 mM MgCl2, 1 µg template DNA, and 1 µl T7 RNA polymerase (NEB; 500 U/ µl); in a

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volume of 20 µl] were carried out at 37°C for 1 hour. This was followed by 1 µl of

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RNase-free DNase I [2 U/µl] for 30 minutes at 37°C. In vitro synthesized mRNA was

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then gel purified on 6% polyacrylamide 7M urea denaturing gels. After UV shadowing

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and elution of the mRNA with 0.2% SDS, 0.005M EDTA and 0.5M NH4OAc, mRNA

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was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with

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0.2 M NH4OAc and 2.5 volumes of EtOH.

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Isolation of E. coli 30S ribosomal subunits and toeprint assays were done as

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previously described (16). Briefly, 9 µl reactions containing mRNA (44nM) with a 32P-

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labelled primer annealed to the 3’ end, 30S ribosomal subunits, tRNAfMet (1:10:20 ratio)

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and 1XSB [60mM NH4Cl, 10mM Tris-OAc (pH 7.4), 10mM MgOAc, 6mM beta-

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mercaptoethanol] were incubated at 37°C for 30 minutes or the indicated times.

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Reactions were placed on ice and 1µl of reverse transcriptase (1 U/µl) was added

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followed by a 37°C incubation for 10 minutes. Reactions were precipitated with 40 µl of

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0.3M NaOAc and 2.5 volumes of EtOH for at least 2.5 hours at -20°C and

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electrophoresed on 6% polyacrylamide (7M urea) gels. Dideoxy sequencing reactions

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mapped toeprint signals to the +16 position of mRNA relative to the first base of the start

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codon (+1). Toeprint signals were quantified with a Molecular Dynamics

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PhosphorImager (Storm 800) and expressed as relative toeprint complexes (RTC)

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[toeprint pixel value/(toeprint pixel value + full-length pixel value)] or [toeprint pixel

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value/(toeprint pixel value + full-length pixel value + upstream signal pixel value)] for

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aroL RTCs. Comparisons of RTCs between different mRNAs were made from reactions

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electrophoresed on the same gel.

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Filter binding assays. mRNA used in filter binding assays was synthesized in a 5 µl

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reaction containing 5mM DTT, 2mM NTPs, 14mM MgCl2, 0.5 µl 10X T7 RNA

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polymerase buffer (NEB), 0.05 µg template DNA, 0.35 µl T7 RNA polymerase (NEB;

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500 U/ µl) and 2.3 µl [α-32P]CTP. Filter binding reactions (10 µl) containing

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radiolabeled mRNA (44nM), 30S ribosomal subunits, tRNAfMet (1:10:20 ratio) and 1XSB

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were incubated for various amounts of time at 37°C. Reaction mixtures were then diluted

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to 500 µl with chilled 1XSB and filtered through a nitrocellulose membrane (0.45-µm

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pore size) in a Mini-Fold slot blot manifold (Schleicher and Schuell) followed by the

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washing of each well with 3 ml of 1XSB. Membranes were then dried at room

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temperature and samples cross-linked to the membranes by UV light (Fisher Scientific,

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FB-UVXL-1000). The amount of complex bound to the membrane was quantified with a

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Molecular Dynamics PhosphorImager (Storm 800); standard curves converted pixel

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values to picomoles, as previously described (4), and data expressed as picomoles of

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mRNA bound.

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Results

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All base substitutions in the aroL, pncB and cysJ genes were made via sitedirected mutagenesis and maintain the natural amino acid sequence; putative “down”

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mutations decrease the downstream adenine content whereas putative “up” mutations

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increase the downstream adenine content (Table 1). The effects of the mutations on gene

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expression were assessed by translational fusions of aroL, pncB and cysJ gene fragments

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to an E. coli lacZ reporter gene followed by β-galactosidase assays. Toeprint and filter

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binding assays were used to compare the rate of ternary complex formation, defined here

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as the amount of complex formed (ribosomes/tRNA/mRNA) over time, for aroL, pncB

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and cysJ mRNAs with or without mutations that alter the downstream adenine content

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(Table 1). In toeprint assays, variations in a specific RTC might be observed between

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different assays but the correlation between wild type and mutant binding strengths were

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consistent and reproducible. These assays were performed in an effort to correlate the in

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vitro ribosome binding strength of aroL, pncB and cysJ mRNAs with the in vivo

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expression data.

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Downstream adenines of aroL

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Transcription of the aroL gene, encoding shikimate kinase II, results in an mRNA

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with a 124 nucleotide leader containing an optimally spaced (7-9 nucleotides), canonical

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SD sequence (5). Mutations in aroL’s coding sequence altering the downstream adenine

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content are shown in Table 1.

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i. The effect of aroL downstream adenines on in vivo expression

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Comparisons of β-galactosidase activity between cells containing the wild type (pAaroL.WT) or the putative “down” aroL-lacZ constructs revealed that the A→G

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substitutions in codons two and three (pAaroL.DN1) decreased lacZ expression 72%;

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incorporation of the individual substitutions resulted in a 15% (pAaroL.DN1a) and 44%

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(pAaroL.DN1b) decrease in lacZ expression (Figure 1-A). The putative “up” aroL-lacZ

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construct revealed that the U→A substitution at codon four (pAaroL.UP1) increased lacZ

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expression 38% (Figure 1-A). These results indicate that the downstream adenines

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influence aroL expression significantly, despite the presence of a canonical SD:ASD

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interaction.

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ii. The effect of aroL downstream adenines on in vitro ribosome binding

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Toeprint assays with aroL-lacZ mRNA and 30S subunits revealed a tRNA-

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dependent signal corresponding to position +16 relative to the A (+1) of the AUG start

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codon (Figure 2-A, lanes 7-9). Phosphorimage analysis of toeprint signal intensity

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revealed a 97% reduction from the putative “down” mutant mRNA (Figure 2-B, lane 15)

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compared to wild type (Figure 2-B, lane 14). Incorporation of the U→A putative “up”

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mutation at codon four increased the toeprint signal 278% (Figure 2-B, lane 16)

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compared to wild type (Figure 2-B, lane 14). The toeprint results, when taken with the

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aroL-lacZ expression levels, show a positive correlation between the downstream adenine

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content, in vitro ribosome binding and in vivo expression levels.

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iii. The effect of aroL downstream adenines on the rate and amount of ternary complex

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formed

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Incorporation of the A→G putative “down” mutations at aroL’s second and third

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codons (Figure 3-A, lanes 10-19) resulted in a reduced rate and amount of ternary

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complex formation compared to wild type (Figure 3-A, lanes 1-9), as quantified in Figure

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3-B. Incorporation of the U→A putative “up” mutation at aroL’s fourth codon (Figure 3-

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A, lanes 20-29) resulted in an increased rate and amount of ternary complex formation

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compared to wild type (Figure 3-A, lanes 1-9), as quantified in Figure 3-B. The

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significant amount of toeprint signal at t=0 (Figure 3-A and B) reflects the complex

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formed and cDNA produced during the ten minute incubation time for reverse

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transcriptase. An additional uncharacterized ternary complex signal is observed within

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aroL’s untranslated leader and is noted with an asterisk (*) in Figure 3-A. Because this

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signal occurs upstream to the aroL toeprint signal (arrow, Figure 3-A), it was included

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with the full length signal for phosphorimage quantification of RTC values in Figure 3-B.

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Calculation of RTC values without the additional band (*) resulted in higher RTC values

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but the line slopes, relative to each other, were basically unchanged (data not shown).

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In order to get a more accurate assessment of complex formation at earlier time

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points, filter binding assays were used. Filter binding assays measure complex formation

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based on the amount of mRNA bound by ribosomes when filtered through a

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nitrocellulose membrane. Incorporation of the A→G putative “down” mutations at

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aroL’s second and third codons reduced the rate and amount of ternary complex formed,

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whereas the U→A putative “up” mutation at codon four increased the amount of ternary

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complex formed compared to wild type (Figure 3-C). Ternary complex formation with

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the “up” mutant demonstrated a similar rate to the wild type during the first 60 seconds of

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incubation, after which the amount of product formed continued to increase with the “up”

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mutant but not with the wild type (Figure 3-C). The toeprint and filter binding results

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provide evidence that downstream adenines enhance the rate and/or amount of ternary

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complex formation with aroL mRNA.

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Downstream adenines of pncB Transcription of the pncB gene, encoding a nicotinic acid

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phosphoribosyltransferase, results in an mRNA with a 58 nucleotide untranslated leader

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containing a canonical SD sequence with suboptimal spacing (12 nucleotides) from the

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start codon (40) and identical second and third codons to aroL. Mutations in pncB’s

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coding sequence altering the downstream adenine content are shown in Table 1.

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i. The effect of pncB downstream adenines on in vivo expression

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Comparisons of β-galactosidase activities between cells containing the wild type

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(pApncB.WT) or the putative “down” pncB-lacZ constructs revealed that the A→G

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substitutions in codons two and three (pApncB.DN1) decreased lacZ expression 99.9%;

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incorporation of the individual substitutions resulted in an 89% (pApncB.DN1a) and 92%

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(pApncB.DN1b) decrease in lacZ expression (Figure 1-B). The putative “up” pncB-lacZ

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construct revealed that the U→A substitutions in codons five and six (pApncB.UP1)

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unexpectedly decreased lacZ expression 67% (Figure 1-B).

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To determine if altering the pncB downstream adenine content has a similar effect

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on expression irrespective of the untranslated leader sequence, analogous pncB-lacZ

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coding sequence fusions were placed downstream of the E. coli lacZ untranslated leader

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containing a canonical SD sequence with optimal spacing (7 nucleotides) from the start

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codon. Putative “down” mutations in codons two and three of the lac-leadered pncB-lacZ

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mRNA reduced gene expression 87% relative to wild type (data not shown). These

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results indicate that the properly spaced canonical SD sequence of the lac leader could

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not fully compensate for the loss of downstream adenines.

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ii. The effect of pncB downstream adenines on in vitro ribosome binding

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Toeprint assays with pncB-lacZ mRNA and 30S subunits revealed a tRNAdependent signal corresponding to position +16 relative to the A (+1) of the AUG start

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codon (Figure 2-A, lanes 1-3). pncB-lacZ mRNA containing putative “down” mutations

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at codon three (Figure 2-B, lane 4) or codons two and three (Figure 2-B, lane 5) nearly

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eliminated ribosome binding (toeprint signal reduced >95%) compared to wild type

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(Figure 2-B, lane 3). Incorporation of the putative “up” mutations at codons five and six

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resulted in a 62% reduction in toeprint signal intensity (Figure 2-B, lane 6) compared to

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wild type (Figure 2-B, lane 3). The toeprint results, when taken with the pncB-lacZ

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expression levels, show a positive correlation between in vitro ribosome binding and the

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in vivo expression levels.

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iii. The effect of pncB downstream adenines on the rate and amount of ternary complex

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formed

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Incorporation of the A→G putative “down” mutation at pncB’s third codon

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(Figure 4-A, lanes 9-16) resulted in a dramatically reduced rate and amount of ternary

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complex formation when compared to wild type (Figure 4-A, lanes 1-8), as quantified in

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Figure 4-B. Bands observed in the absence of tRNA and ribosomes are artifacts likely

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due to structures formed within the mRNA. When analyzed by filter binding assays,

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incorporation of the A→G putative “down” mutation at pncB’s third codon resulted in a

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reduced rate and amount of ternary complex formation compared to wild type (Figure 4-

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C). The toeprint and filter binding results provide evidence that downstream adenines

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contribute to the rate and amount of ternary complex formation with pncB mRNA.

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Downstream adenines of cysJ The E. coli cysJ gene, encoding a NADPH-sulfite reductase flavoprotein

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component (22), has different second and third codons from aroL and pncB. Cloning a

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DNA fragment encoding the cysJ untranslated leader and a fragment of cysJ coding

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sequence into a multicopy plasmid caused a dramatic reduction in plasmid copy number

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(data not shown); therefore, cysJ-lacZ coding sequence fusions were placed downstream

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of the 38 nucleotide lacZ untranslated leader containing a canonical SD sequence with

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optimal spacing (7-9 nucleotides) from the cysJ start codon. Mutations in cysJ’s coding

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sequence altering the downstream adenine content are shown in Table 1.

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i. The effect of cysJ downstream adenines on in vivo expression

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Comparisons of β-galactosidase activities between cells containing the wild type

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(pAZcysJ.WT) or the putative “down” (pAZcysJ.DN1) cysJ-lacZ construct revealed that

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the third codon A→G base substitution resulted in a 77% decrease in lacZ expression

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(Figure 1-C). The putative “up” cysJ-lacZ constructs revealed that the G→A

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substitutions in codons two and four (pAZcysJ.UP1) increased lacZ expression 525%;

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incorporation of the individual substitutions resulted in a 174% (pAZcysJ.UP1a) and

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225% (pAZcysJ.UP1b) increase in expression (Figure 1-C).

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ii. The effect of cysJ downstream adenines on in vitro ribosome binding

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Toeprint assays with cysJ-lacZ mRNA and 30S subunits revealed a tRNA-

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dependent signal corresponding to position +16 relative to the A (+1) of the AUG start

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codon (Figure 2-A, lanes 4-6). Phosphorimage analysis of toeprint signal intensity

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revealed an 86% reduction from the putative “down” mutant (Figure 2-B, lane 10)

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compared to wild type (Figure 2-B, lane 9). Incorporation of putative “up” mutations at

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codons two and four resulted in a 202% increase in toeprint signal intensity (Figure 2-B,

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lane 11) compared to wild type (Figure 2-B, lane 9). The toeprint results, when taken

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with the cysJ-lacZ expression levels, show a strong correlation between the downstream

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adenine content, in vitro ribosome binding and in vivo expression levels.

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iii. The effect of cysJ downstream adenines on the rate and amount of ternary complex

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formed

304

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Incorporation of the A→G putative “down” mutation at cysJ’s third codon (Figure 5-A, lanes 10-18) resulted in a reduced rate and amount of ternary complex formation

306

compared to wild type (Figure 5-A, lanes 1-9), as quantified in Figure 5-B. Incorporation

307

of the G→A putative “up” mutations at cysJ’s second and fourth codons (Figure 5-A,

308

lanes 19-27) resulted in an increased rate and amount of ternary complex formation

309

compared to wild type (Figure 5-A, lanes 1-9), as quantified in Figure 5-B.

310

Phosphorimage analysis of the full length, run-off signal yielded consistently high pixel

311

values, resulting in low RTC values; however, the rate comparisons between mutant and

312

WT mRNAs still correlated well with results visualized by autoradiography. When

313

analyzed by filter binding assays, incorporation of the A→G putative “down” mutation at

314

cysJ’s third codon resulted in a similar rate and amount of ternary complex formation as

315

with wild type mRNA during the first three minutes of incubation; after three minutes,

316

the rate and amount of ternary complex formed was reduced compared to wild type

317

(Figure 5-C). Incorporation of the G→A putative “up” mutations at cysJ’s second and

318

fourth codons resulted in an increased rate and amount of ternary complex formation

319

compared to wild type, with the most significant difference in rate occurring within the

320

initial three minutes of binding (Figure 5-C). The toeprint and filter binding results

E C

C A

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T P

305

321

provide evidence that downstream adenines enhance the rate and amount of ternary

322

complex formed with cysJ mRNA.

T P

E C

C A

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Discussion

323 324

The correlation of adenine-rich regions with efficient translation in E. coli was first noted by Dreyfus (8), with later studies reporting stimulatory effects on translation

326

by the addition of adenines (3, 16, 30). We report here that adenines occurring naturally

327

downstream of the initiation codon can contribute significantly to ribosome binding and

328

expression in E. coli. Our data show that downstream adenines can exert major effects

329

on expression even in the presence of canonical SD sequences. Demonstration that

330

downstream nucleotides can function as major effectors of expression is especially

331

interesting in light of a recent report (2) that non-SD led genes, including leaderless

332

mRNAs, are as common as SD-led genes in prokaryotes. The absence of a SD sequence

333

requires that other sequence or structural features of the mRNA help direct the ribosome

334

to the correct initiation site. From work described here, the position and number of

335

downstream adenines may contribute to the ribosome binding strength of mRNAs with,

336

and possibly without, canonical SD sequences.

337

D E

T P

E C

C A

Our experiments investigate the influence of naturally occurring downstream

338

adenines on in vitro ribosome binding and in vivo expression for the E. coli aroL, pncB

339

and cysJ mRNAs. When compared to the wild type mRNAs, the putative “down”

340

mutations significantly decreased in vitro ribosome binding and in vivo expression;

341

putative “up” mutations in aroL and cysJ significantly increased in vitro ribosome

342

binding and in vivo expression. These data are consistent with earlier reports whereby A-

343

rich sequences added immediately downstream of the start codon stimulated expression

344

from several natural and heterologous genes in E.coli (16, 3). While the pncB “down”

345

mutations demonstrate clearly that adenines in codons two and three are important for

18

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325

346

expression, it is unclear why the putative “up” mutations in codons five and six did not

347

increase expression; possible explanations are that the substitutions reside too far from

348

the start codon (i.e., compared to codons two and four for cysJ and aroL ”up” mutations)

349

or the mutations resulted in a secondary structure that reduced access of a ribosome to the

350

initiation region.

351

Downstream adenines affect rate and amount of ternary complex formed.

352

D E

Using toeprint and filter binding assays as two independent approaches to

estimate ribosome binding, we observed a strong correlation between the downstream

354

adenine content, in vivo expression and the rate and amount of in vitro ribosome binding,

355

suggesting strongly that the variations in expression were mediated through the observed

356

effects on ribosome binding. Toeprint assays revealed also that aroL, pncB and cysJ

357

ternary complexes, once formed, did not dissociate in the presence of a competitor

358

mRNA (data not shown), in agreement with a previous report that ternary complexes are

359

stable and irreversible in the presence of competitor mRNA (31). Therefore, downstream

360

adenines appear to influence expression through their affect on mRNA-ribosome

361

association and not by preventing ternary complex dissociation.

362

Possible contributions of downstream adenines to ribosome binding and expression

363

Comparison of the codons resulting from aroL, pncB and cysJ mutagenesis to an

364

E. coli genomic codon usage table (GenBank) suggests that the effects on expression are

365

not due to the introduction of rare codons or to low tRNA availability. Also,

366

conservation of the wild type amino acid sequence ensured that expression differences

367

from the mutant constructs were not the result of an altered peptide sequence.

368

Furthermore, the strong correlation between in vitro ribosome binding and in vivo

E C

C A

19

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T P

353

369

expression, for both wild type and mutants, suggests the downstream adenines exert their

370

affect at initiation rather than elongation.

371

It is possible that the adenine effects seen here are due to structure within the TIR. It has been reported that secondary structure within the TIR may inhibit expression,

373

whereas a more open TIR with less structure allows increased expression (6, 7). In an

374

effort to explore the possible contribution of structure to our results, various lengths of

375

the cysJ-, pncB- and aroL-lacZ mRNAs, with or without mutations, were subjected to

376

MFOLD analysis (43) for prediction of mRNA secondary structures by energy

377

minimization. While slight variations in the predicted structures resulted from the

378

downstream base substitutions, a general correlation of more closed or open structures

379

with respective decreases or increases in expression and ribosome binding was not

380

observed (data not shown). In addition, comparing the signals from reverse transcriptase

381

pausing or terminating at inhibitory secondary structures during toeprint assays does not

382

suggest significant differences in structure between wild type and mutant mRNAs

383

(Figures 3-5). Another possibility involving structure is the formation of adenine-rich

384

pseudoknots, described previously as high-affinity RNA ligands to 30S subunits and

385

ribosomal protein S1 (23). While a contribution by downstream adenines to a stimulatory

386

secondary structure has not been discounted, we do not have any data to support this

387

model.

388

D E

T P

E C

C A

It is also possible that downstream adenines, perhaps in association with

389

pyrimidines (16), provide a region within the RBS that is recognized specifically by

390

ribosome-associated proteins that promote initiation complex formation. For example,

391

the 30S subunit protein S1 is a strong RNA binding protein reported to concentrate

20

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372

mRNA near the ribosome decoding site (36, 35, 1). Previous studies revealed the binding

393

of S1 to various nucleotide motifs within the untranslated leader including an AU-rich

394

omega sequence (10, 1, 14, 15), a CAU-rich omega-like sequence (38) and a (CAA)n

395

repeat (38). From these data it is conceivable that S1 could bind downstream A-rich

396

regions for a more efficient delivery of mRNA to the ribosome decoding site; however,

397

there has been no direct evidence that S1 binds specific nucleotide motifs downstream of

398

the start codon. Interaction between downstream A-rich regions and 16S rRNA might

399

also contribute to mRNA-ribosome association. In possible support of this, poly (A)

400

RNA formed crosslinks to 16S rRNA nucleotides 1394-1399, located adjacent to the

401

ribosomal P-site (34).

402

D E

T P

E C

Crystal structures of ribosome:mRNA complexes provide evidence that mRNA

403

positions -3 to +10 (with the A of the AUG start codon as +1) are wrapped closely around

404

the neck of the 30S subunit, with most ribosome contacts involving the mRNA backbone

405

rather than its bases (41). However, these crystal structures represent stable complexes

406

and lend little information on interactions occurring during ribosome loading that lead to

407

a stable complex.

408

C A

Recent experiments from our lab also suggest contact between a natural adenine-

409

rich sequence of a leaderless mRNA and proteins associated with E. coli 30S subunits. In

410

this work, leaderless mRNA encoded by the cI gene of bacteriophage lambda, with a

411

photoactivatable 4-thio uridine in the AUG start codon, has been crosslinked to 30S

412

subunit proteins (J. E. Brock and G. R. Janssen, unpublished data). Crosslinking is

413

inhibited, however, by a competitor cI mRNA that lacked a start codon but contains the

414

natural adenine-rich sequence immediately downstream of the start codon position. Also,

21

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392

415

the same cI competitor mRNA inhibits ribosome binding to wild type cI mRNA in

416

toeprint assays suggesting that the adenines contribute to an interaction between mRNA

417

and components of the ribosome that facilitate ternary complex formation. Additional

418

support for downstream adenine stimulation of leaderless mRNA translation in E. coli is

419

provided by a report (16) where addition of CA multimers increased expression from

420

leaderless lacZ, gusA and neo mRNAs.

421

Implications and application of downstream adenines for expression

T P

Although the mechanistic details for adenine-enhanced ribosome binding and

423

expression remain to be determined, their ability to stimulate translation from leaderless

424

mRNA (16) indicates that an untranslated leader or SD:ASD interaction is not required.

425

Even in the presence of a canonical SD:ASD interaction, downstream adenines

426

significantly influenced ribosome binding and expression from aroL, pncB and cysJ

427

mRNAs. The first five codons of coding sequence are protected by a ribosome during

428

formation of an initiation complex and thereby represent potential contacts that could

429

contribute to an mRNA’s ribosome binding strength; however, constraints imposed by

430

the encoded amino acid sequence has made it difficult to consider sequence-specific

431

translation enhancers in this region of the mRNA. Genetic code degeneracy and “wobble

432

position” variability, however, could allow for a bias towards adenines within the first

433

few codons while imposing minimal constraints on the encoded amino acids.

434

Downstream adenines, possibly in conjunction with the start codon or other features of

435

the mRNA, might present a recognition motif (sequence and/or structural) to a

436

component of the translational machinery and contribute importantly to the ribosome

E C

C A

22

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422

D E

437

binding strength, with the number and position of adenines allowing for “fine-tuning” of

438

expression.

439

If downstream adenines merely provide an open structure for access of the start codon to ribosomes, then one might expect adenines to stimulate expression in a variety

441

of translation systems; however, addition of downstream adenines to a neo reporter gene

442

did not increase expression from the G+C-rich, gram-positive Streptomyces lividans (J.

443

M. Day and G. R. Janssen, unpublished data). The possibility that adenine stimulation

444

might not occur in G+C-rich organisms argues that the enhanced ribosome binding and

445

expression is at least partially based on sequence and suggests that adenine stimulation of

446

translation might be limited to E. coli and related organisms. Using crosslinking

447

techniques to identify downstream adenine/ribosome interactions, along with further

448

structural studies, may provide evidence to distinguish between sequence and structural

449

components of adenine stimulation.

450

D E

T P

E C

C A

Characterization of the adenine effect might allow also for simple engineering of

451

expression levels, up or down, by increasing or decreasing downstream adenines.

452

Identification of mRNA features contributing to ribosome binding and translation, from

453

coding sequences with or without SD sequences (2), is essential to understanding these

454

important stages of gene expression in E. coli and other organisms.

23

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440

References

455 456

1. Boni, I.V., D. M. Isaeva, M. L. Musychenko, and N. V. Tzareva. 1991.

457

Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1.

458

Nucleic Acids Res. 19:155-162.

459

D E

2. Chang, B., S. Halgamuge, and S. Tang. 2006. Analysis of SD sequences in

460

completed microbial genomes: Non-SD-led genes are as common as SD-led

461

genes. Gene 373:90-99.

T P

3. Chen, H., L. Pomeroy-Cloney, M. Bjerknes, J. Tam, and E. Jay. 1994. The

463

influence of adenine rich motifs in the 3’ portion of the ribosome binding site on

464

human IFN-γ gene expression in Escherichia coli. J. Mol. Biol. 240:20-27.

465 466 467 468 469 470 471

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4. Day, M. J., and G. R. Janssen. 2004. Isolation and characterization of ribosomes and translational initiation factors from the gram-positive soil bacterium

C A

Streptomyces lividans. J. Bacteriol. 186:6864-6875.

5. DeFeyter, R. C., B. E. Davidson, and J. Pittard. 1986. Nucleotide sequence of the transcription unit containing the aroL and aroM genes from Escherichia coli K-12. J. Bacteriol. 165:233-239.

6. de Smit, M.H., and J. van Duin. 1990a. Control of prokaryotic translational

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initiation by mRNA secondary structure. Proc. Nucleic Acid Res. Mol. Biol. 38:1-

473

35.

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7. de Smit, M.H., and J. van Duin. 1990b. Secondary structure of the ribosome

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binding site determines translational efficiency: a quantitative analysis. Proc. Natl.

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Acad. Sci. USA 87:7668-7672.

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477 478 479

8. Dreyfus, M. 1988. What constitutes the signal for the initiation of protein synthesis on Escherichia coli mRNAs? J. Mol. Biol. 204(1):79-94. 9. Etchegaray, J.P., and M. Inouye. 1999. Translational enhancement by an

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element downstream of the initiation codon in Escherichia coli. J. Biol. Chem.

481

274:10079-10085.

10. Gallie, D.R., D. E. Sleat, J. W. Wyatts, P. C. Turner, and T. M. A. Wilson.

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1987. The 5 prime leader sequence of tobacco mosaic virus RNA enhances the

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expression of foreign gene transcripts in vitro and in vivo. Nucleic Acids Res.

485

15:3257-3273.

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11. Gualerzi, C. O., L. Brandi, E. Caserta, A. La Teana, R. Spurio, J. Tomsic,

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and C. L. Pon. 2000. Translation initiation in bacteria, p. 477-494. In R. A.

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Garrett, S. R. Douthwaite, A. Liljas, A. T. Matheson, P. B. Moore, and H. F.

489 490 491 492 493 494

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Noller (ed.), The ribosome: structure, function, antibiotics, and cellular interactions. ASM Press, Washington, D.C.

12. Ivanov, I.G., R. Alexandrova, B. Dragulev, D. Leclerc, A. Saraffova, V. Maximova, and M. G. Abouhaidar. 1992. Efficiency of the 5’-terminal sequence (omega) of tobacco mosaic virus RNA for the initiation of eukaryotic gene translation in Escherichia coli. Eur. J. Biochem. 209:151-156.

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13. Janssen, G.R. 1993. Eubacterial, archaeabacterial, and eukaryotic genes that

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encode leaderless mRNA, p. 59-67. In R. Baltz, G. D. Hegeman, and P. L.

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Skatrud (ed.), Industrial microorganisms: basic and applied molecular genetics.

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American Society for Microbiology, Washington, D.C.

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14. Komarova, A. V., L. S. Tchufistova, E. V. Supina, and I. V. Boni. 2002.

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Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno

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sequence on translation. RNA 8:1137-1147.

502

15. Komarova, A. V., L. S. Tchufistova, M. Dreyfus, and I. V. Boni. 2005. AU-

D E

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rich sequences within 5’ untranslated leaders enhance translation and stabilize

504

mRNA in Escherichia coli. J. Bacteriol. 187:1344-1349.

505

16. Martin-Farmer, J. A., and G. R. Janssen. 1999. A downstream CA repeat sequence increases translation from leadered and unleadered mRNA in

507

Escherichia coli. Mol. Microbiol. 31:1025-1038.

508 509 510 511 512 513 514 515 516 517

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17. Miller, J. 1992. In A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

18. Moll, I., S. Grill, C. O. Gualerzi, and U. Blasi. 2002. Leaderless mRNAs in

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bacteria: surprises in ribosomal recruitment and translational control. Mol. Microbiol. 43:239-246.

19. O’Conner, M., T. Asai, C. L. Squires, and A. E. Dahlberg. 1999. Enhancement of translation by the downstream box does not involve base pairing of mRNA with the penultimate stem sequence of 16S rRNA. Proc. Natl. Acad. Sci. USA 96:8973-8978. 20. O’Donnell, S. M., and G. R. Janssen. 2001. The initiation codon effects

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ribosome binding and translational efficiency in Escherichia coli of cI mRNA

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with or without the 5’ untranslated leader. J. Bacteriol. 183:1277-1283.

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21. Olins, P.O., and H. S. Rangwala. 1989. A novel sequence element derived from

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bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in E.

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coli. J. Mol. Biol. 264:16973-16976.

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22. Ostrowski, J., and N. M. Kredich. 1989. Molecular characterization of the cysJIH promoters of Salmonella typhimurium and Escherichia coli: regulation by

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cysB protein and N-acetyl-L-serine. J. Bacteriol. 171:130-140.

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23. Ringquist, S., T. Jones, E. E. Snyder, T. Gibson, I. Boni, and L. Gold. 1995.

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High-affinity RNA ligands to Escherichia coli ribosomes and ribosomal protein

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S1: comparison of natural and unnatural binding sites. Biochemistry 34:3640-

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3648.

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24. Rudd, K. E., and T. D. Schneider. 1992. Compilation of E. coli ribosome

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binding sites, p. 17.19-17.46. In J. Miller (ed.), A Short Course in Bacterial

532 533 534 535 536

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Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

25. Sambrook, J., E. F. Fritsch, T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

26. Sato, T., M. Terabe, H. Watanabe, T. Gojobori, C. Hori-Takemoto, and K.

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Miura. 2001. Codon and base biases after the initiation codon of the open reading

538

frames in the Escherichia coli genome and their influence on the translation

539

efficiency. J. Biochem. 129:851-860.

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524

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27. Scherer G. F. E., M. D. Walkinshaw, S. Arnott, and D. J. Morr. 1980. The

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ribosome-binding sites recognized by E. coli ribosomes have regions with signal

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character in both the leader and protein coding segments. Nucleic Acids Res.

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8:3895-3907.

544 545

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28. Schleif, R. 1972. Fine-structure deletion map of the Escherichia coli L-arabinose operon. Proc. Natl. Acad. Sci. USA 11:3479-84.

29. Shine, J., and L. Dalgarno. 1974. The 3’-terminal sequence of Escherichia coli

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16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding

548

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549 550 551 552 553 554 555 556 557 558

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30. Slovek, L. E. 1999. M.S. thesis. Miami University, Oxford. Ribosomal binding and enhanced translation of CA-containing mRNA in Escherichia coli. 31. Spedding, G., T. Gluick, and D. Draper. 1993. Ribosome initiation complex

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formation with the pseudoknotted α operon messenger RNA. J. Mol. Biol. 229:609-622.

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34. Stiege, W., K. Stade, D. Schuller, and R. Brimacombe. 1988. Covalent cross-

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linking of poly (A) to Escherichia coli ribosomes, and localization of the cross-

561

link site within the 16S rRNA. Nucleic Acids Res. 16:2369-2388.

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35. Subramanian, A. R. 1984. Structure and function of the largest Escherichia coli ribosomal protein. TIBS 9:491-494. 36. Suryanarayana, T., and A. R. Subramanian. 1983. An essential function of

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ribosomal protein S1 in messenger ribonucleic acid translation. Biochemistry

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22:2715-2719.

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37. Thanaraj, T.A., and M. W. Pandit. 1989. An additional ribosome binding site on mRNA of highly expressed genes and a bifunctional site on the colicin

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translation initiation. Nucleic Acid Res. 18:2973-2985.

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38. Tzareva, N.V., V. I. Makhno, and I. V. Boni. 1994. Ribosome-messenger

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recognition in the absence of the Shine-Dalgarno interactions. FEBS Lett.

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337:189-194.

574 575 576 577 578 579 580

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39. Wade, H. E., and H. K. Robinson. 1966. Magnesium ion-independent ribonucleic acid depolymerases in bacteria. Biochem. J. 101:467-479.

40. Wubbolts, M. G., P. Terpstra, J. B. van Beilen, J. Kingma, H. A. R. Meesters, and B. Witholt. 1990. Variation of cofactor levels in Escherichia coli. J. Biol. Chem. 265:17665-17672.

41. Yusupova, G. Z., M. M. Yusupova, J. H. D. Cate, H. F. Noller. 2001. The path of messenger RNA through the ribosome. Cell 106:233-241.

581

42. Zhang, J., and M. P. Deutscher. 1991. A uridine rich sequence required for

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583 584

43. Zuker, M. 22 Aug 2006. MFOLD version: 5.a (17:13). Rensselaer Polytechnic Institute. [Online.] http://bioweb.pasteur.fr/cgi-bin/seqanal/mfold.pl

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568

618 619 620 621 622

Table 1. DNA sequence of gene fragments, with or without mutations, from the aroL, pncB and cysJ genes. Plasmid a

Sequence b

aroL-lacZ fusions pAaroL.WT: 5’…tggggaaaacccacgATG pAaroL.DN1: …ATG pAaroL.DN1a: …ATG pAaroL.DN1b: …ATG pAaroL.UP1: …ATG Amino Acid: M

ACA ACG ACG ACA ACA T

pncB-lacZ fusions pApncB.WT: 5’…caggatactgcgcacctATG pApncB.DN1: …ATG pApncB.DN1a: …ATG pApncB.DN1b: …ATG pApncB.UP1: …ATG Amino Acid: M

cysJ-lacZ fusions pAZcysJ.WT: 5’…caggaaacagccATG pAZcysJ.DN1: …ATG pAZcysJ.UP1: …ATG pAZcysJ.UP1a: …ATG pAZcysJ.UP1b: …ATG Amino Acid: M

a

CAA CAG CAA CAG CAA Q

ACA ACG ACG ACA ACA T

ACG ACG ACA ACA ACG T

CCT CCT CCT CCT CCA P

CAA CAG CAA CAG CAA Q

ACA ACG ACA ACA ACA T

TTC TTC TTC TTC TTC F

CAG CAG CAA CAG CAA Q

CTT CTT CTT CTT CTT P

TTT TTT TTT TTT TTT F

GCT GCT GCT GCT GCA A

GTC GTC GTC GTC GTC V

CTG…3’ CTG… CTG… CTG… CTG… P

TCT CCT…3’ TCT CCT… TCT CCT… TCT CCT… TCA CCT… S P

D E

T P

CCA…3’ CCA… CCA… CCA… CCA… P

E C

The pA-series of plasmid constructs contain the genes’ natural untranslated

C A

leader while the pAZ-series contain the lac untranslated leader. b

Lower case letters represent a portion of the untranslated leader and is shown only

for the wild type (WT) constructs, but is present also in each mutant below it. The lower case, underlined sequences indicate the presumed SD sequence. The upper case ATG identifies the translational start site for the aroL, pncB

623

and cysJ coding sequences. The single letter code corresponding to the

624

encoded amino acids is given below the sequence. The upper case, underlined,

625

bold-faced nucleotides indicate base substitutions within the coding region to

626

create putative “down” or “up” mutations.

30

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585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617

Figure Legends

627

Figure 1. The effect of aroL, pncB and cysJ downstream adenines on expression in

629

vivo. β-galactosidase activities from aroL-, pncB- and cysJ-lacZ fusions are compared

630

between cells containing plasmids with or without mutations altering downstream

631

adenine content (see Table 1). A) Plasmid-free cells and cells containing plasmids with

632

the following aroL-lacZ fusions: pAaroL.WT, 100%; pAaroL.DN1, 28%; pAaroL.DN1a,

633

85%; pAaroL.DN1b, 56%; pAaroL.UP1, 138%. B) Plasmid-free cells and cells

634

containing plasmids with pncB-lacZ fusions: pApncB.WT, 100%; pApncB.DN1, 0.1%;

635

pApncB.DN1a, 11%; pApncB.DN1b, 8%; pApncB.UP1, 33%. C) Plasmid-free cells and

636

cells containing plasmids with cysJ-lacZ fusions: pAZcysJ.WT, 100%; pAZcysJ.DN1,

637

23%; pAZcysJ.Up1, 625%; pAZcysJ.UP1a, 274%; pAZcysJ.UP1b, 325%.

638

Figure 2. The effects of aroL, pncB and cysJ downstream adenines on ribosome

639

binding in vitro. Toeprint assays with aroL-, pncB- and cysJ-lacZ mRNAs and 30S

640

subunits compare ribosome binding to mRNAs with or without mutations altering

641

downstream adenine content (see Table 1). The closed arrows indicate the ternary

642

complex-dependent toeprint signals at position +16 relative to the A (+1) of the AUG

643

start codons that result from bound 30S subunits and the asterisk (*) in (B) indicates an

644

uncharacterized ternary complex-dependent signal within the aroL upstream UTR.

645

Bands observed in the absence of tRNA and ribosomes are artifacts likely due to

646

structures formed within the mRNA. A) Lanes: GATC, dideoxy DNA sequence ladder

647

for the pncB template, with the ATG initiation triplet boxed; 1-3, pncB-lacZ WT mRNA;

648

4-6, cysJ-lacZ WT mRNA; 7-9, aroL-lacZ WT mRNA. B) Lanes 1-6 utilize pncB-lacZ

649

mRNAs made from the following templates: (1-3) pApncB.WT, (4) pApncB.DN1b, (5)

D E

T P

E C

C A

31

Downloaded from jb.asm.org at Penn State Univ on February 8, 2008

628

pApncB.DN1, (6) pApncB.UP1. Lanes 7-11 utilize cysJ-lacZ mRNAs made from the

651

following templates: (7-9) pAZcysJ.WT, (10) pAZcysJ.DN1, (11) pAZcysJ.UP1. Lanes

652

12-16 utilize aroL-lacZ mRNAs made from the following templates: (12-14)

653

pAaroL.WT, (13) pAaroL.DN1, (14) pAaroL.UP1.

654

Figure 3. The effects of aroL downstream adenines on the rate and amount of

655

ternary complex formed. Toeprint and filter binding assays compare ternary complex

656

formation on aroL-lacZ mRNAs with or without mutations altering downstream adenine

657

content. A) In toeprint assays, 30S subunits were incubated with tRNAfMet and aroL-lacZ

658

mRNA for increasing times (minutes). Lanes 1-29 utilize aroL-lacZ mRNAs made from

659

the following templates: (1-9) pAaroL.WT, (10-19) pAaroL.DN1, (20-29) pAaroL.UP1.

660

The closed arrow indicates the position of the ternary complex-dependent toeprint signals

661

at +16 and the asterisk (*) indicates an uncharacterized ternary complex-dependent signal

662

within the upstream UTR. B) The relative toeprint complex (RTC) formed with time was

663

quantified and the data averaged from three independent assays are plotted: ν, aroL-lacZ

664

WT mRNA (pAaroL.WT); Ο, aroL-lacZ “down” mutant mRNA (pAaroL.DN1); ∆, aroL-

665

lacZ “up” mutant mRNA (pAaroL.UP1). C) In filter binding assays, 30S subunits were

666

incubated with tRNAfMet and aroL-lacZ mRNA for increasing time and the amount of

667

bound RNA, averaged from three independent assays, is plotted: ν, aroL-lacZ WT

668

mRNA (pAaroL.WT); Ο, aroL-lacZ “down” mutant mRNA (pAaroL.DN1); ∆, aroL-lacZ

669

“up” mutant mRNA (pAaroL.UP1).

670

Figure 4. The effects of pncB downstream adenines on the rate and amount of

671

ternary complex formed. Toeprint and filter binding assays compare ternary complex

672

formation on pncB-lacZ mRNAs with or without mutations altering downstream adenine

D E

T P

E C

C A

32

Downloaded from jb.asm.org at Penn State Univ on February 8, 2008

650

content. A) In toeprint assays, 30S subunits were incubated with tRNAfMet and pncB-

674

lacZ mRNA for increasing times (minutes). Lanes 1-16 utilize pncB-lacZ mRNAs made

675

from the following templates: (1-8) pApncB.WT, (9-16) pApncB.DN1b. The arrow

676

indicates the position of the ternary complex-dependent toeprint signals. B) The relative

677

toeprint complex (RTC) formed with time was quantified and the data averaged from

678

three independent assays are plotted: ν, pncB-lacZ WT mRNA (pApncB.WT); Ο, pncB-

679

lacZ “down” mutant mRNA (pApncB.DN1b). C) In filter binding assays, 30S subunits

680

were incubated with tRNAfMet and pncB-lacZ mRNA for increasing time and the amount

681

of bound RNA, averaged from three independent assays, is plotted: ν, pncB-lacZ WT

682

mRNA (pApncB.WT); Ο, pncB-lacZ “down” mutant mRNA (pApncB.DN1b).

683

Figure 5. The effects of cysJ downstream adenines on the rate and amount of

684

ternary complex formed. Toeprint and filter binding assays compare ternary complex

685

formation on cysJ-lacZ mRNAs with or without mutations altering downstream adenine

686

content. A) In toeprint assays, 30S subunits were incubated with tRNAfMet and cysJ-lacZ

687

mRNA for increasing times (minutes). Lanes 1-27 utilize cysJ-lacZ mRNAs made from

688

the following templates: (1-9) pAZcysJ.WT, (10-18) pAZcysJ.DN1, (19-27)

689

pAZcysJ.UP1. The arrow indicates the position of the ternary complex-dependent

690

toeprint signals. B) The relative toeprint complex (RTC) formed with time was

691

quantified and the data averaged from three independent assays are plotted: ν, cysJ-lacZ

692

WT mRNA (pAZcysJ.WT); Ο, cysJ-lacZ “down” mutant mRNA (pAZcysJ.DN1); ∆,

693

cysJ-lacZ “up” mutant mRNA (pAZcysJ.UP1). C) In filter binding assays, 30S subunits

694

were incubated with tRNAfMet and cysJ-lacZ mRNA for increasing time and the amount

695

of bound RNA, averaged from three independent assays, is plotted: ν, cysJ-lacZ WT

D E

T P

E C

C A

33

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673

696

mRNA (pAZcysJ.WT); Ο, cysJ-lacZ “down” mutant mRNA (pAZcysJ.DN1); ∆, cysJ-

697

lacZ “up” mutant mRNA (pAZcysJ.UP1).

T P

E C

C A

34

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D E

Acknowledgements

698 699 700 701

This work was supported by grant GM065120 from the National Institutes of Health. J.E.B. thanks the Miami University Graduate School for a Graduate Student Achievement Award and the Center for Bioinformatics and Functional Genomics for a

703

summer research fellowship. Special thanks to Eileen Bridge for helpful comments on

704

the manuscript, and Holly Rovito and Michael Day for inspiring discussions and

705

assistance with assay development.

T P

E C

C A

35

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D E

702

ys J.U P1 b

600

pA Zc

700

ys J.U P1 a

C. cysJ D

N 1

pA

1b

1a

P1

N

N

pn cB .U

pn cB .D

pn cB .D

E C pA

pA

pn cB .

0

pA Zc

N 1

pn cB .W T

20

ys J.U P1

sJ .D

pA

pA

40

pA Zc

Zc y

ys J.W T

RF S8 59

% Beta-Galactosidase Activity

P1

1b

N

D

1a

N

1

N

ar oL .U

pA

ar oL .

pA

ar oL .D

pA

W T

ar oL .D

pA

85 9

ar oL .

pA

RF S

% Beta-Galactosidase Activity 20

0

B. pncB

120

100

80

60

500

400

300

200

100

0

T P D E Downloaded from jb.asm.org at Penn State Univ on February 8, 2008

pA

pA Zc

RF S8 59

C A % Beta-Galactosidase Activity

Figure 1. A. aroL 160

140

120

100 80

60

40

Figure 2. A.

pncB

B.

pncB cysJ aroL

cysJ

aroL

D E *

+16

E C

→

Lane C T AG mRNA tRNAfMet 30S

→

12 ++ + - +

3 + + +

4 + + -

5 + +

6 + + +

7 + + -

8 + +

C A

9 + + +

Lane mRNA tRNAfMet 30S

37

1 + + -

2 + +

3 + + +

4 + + +

5 6 ++ ++ ++

7 + + -

8 + +

9 10 11 12 13 14 15 16 + + + + + + + + + + + + - + + + + + + - + + + + \

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T P

A T G

Figure 3. A.

WT

DN1

UP1

T P

E C

→

C A

Lane mRNA tRNAfMet 30S Time

B. 0.9 0.8 0.7 0.6 0.5

1 + + -

2 + +

3 + + +

4 + + +

5 + + +

6 + + +

7 + + +

8 + + +

9 + + +

60 60 0 5 10 15 20 40 60

10 + + -

11 + +

12 + + +

60

60

0

13 14 + + + + + +

15 + + +

16 + + +

5 10 15 15

17 + + +

18 + + +

19 + + +

20 21 + + + - +

20 40 60

60

60

22 + + + 0

23 24 25 26 + + + + + + + + + + + +

27 28 29 + + + + + + + + +

5 10 15 15 20 40 60

C. 0.2

0.18 0.16 0.14 0.12 0.1

0.4

0.08

0.3

0.06

0.2

0.04

0.1

0.02

0

0 0

10

20

30

40

50

60

70

0

Time (minutes)

100

200

300

400

Time (seconds

38

500

600

700

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D E

*

Figure 4. A.

WT

DN1b

B. 0.25 0.2 0.15

0.1

D E

0.05 0 0

10

20

30

40

50

60

70

500

600

700

T P

Time (minutes)

C. 0.16 0.14

E C 0.12

0.1

0.08

Lane mRNA tRNAfMet 30S

1 + +

2 + + -

3 + + +

4 + + +

Time

60 60 0 5

5 + + +

6 + + +

7 + + +

8 + + +

9 10 11 12 + + + + - + + + + - + +

13 14 15 16 + + + + + + + + + + + +

C A 10 20 40 60

60 60 0

5 10 20 40 60

0.06 0.04 0.02

0

0

39

100

200

300

400

Time (seconds)

Downloaded from jb.asm.org at Penn State Univ on February 8, 2008

→

Figure 5.

A.

WT

DN1

UP1

Lane mRNA tRNAfMet 30S Time

B. 0.16 0.14 0.12 0.1 0.08 0.06

1 + + +

2 + + +

3 + + +

4 + + +

5 + + +

6 + + +

7 + + +

8 + +

9 + + -

0

5 10 15 20 40 60 60 60

10 + + +

C A

E C

0

T P

11 + + + 5

12 + + +

13 14 + + + + + +

15 16 + + + + + +

17 18 + + - + + -

10 15 20 40 60 60 60

19 + + +

20 + + +

0

21 22 + + + + + +

23 24 25 + + + + + + + + +

26 27 + + - + + -

5 10 15 20 40 60 60 60

C.

0.12 0.1 0.08 0.06 0.04

0.04

0.02

0.02 0

0 0

10

20

30

40

50

60

70

0

Time (minutes)

100

200

300

400

Time (secon

40

500

600

700

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D E

→