Lecture 1 amazonaws com
BIOB11 Term Test #1 Material [Lecture 18] Lecture 1: Chromosomes, Heredity, Meiosis and Recombination • Diploid: Both members of pair of homologous chromosomes, found in somatic cells • Haploid: Only one member of each pair of homologous chromosomes, produced by meiosis • Allele: Alternative forms of the same gene
• Dominant/ Recessive Genes: Homozygous dominant aka wildtype • Homologous chromosome: Paired chromosomes of diploid cells, that are similar in structure; contains two sister chromatids • Bivalent/Tetrad: Formed during meiosis by junction of two homologous pair • Recombination/ Reciprocal genetic exchange: Genetic rearrangement of genetic material via crossing over or formation of bivalent • Homozygous: two identical alleles of a gene • •
Heterozygous: two different alleles of a gene
Synaptonemal complex: a ladderlike series of parallel threads holding pairing homologous chromosomes in meiosis for recombination to occur. • Nondisjunction: the failure of one or more pairs of homologous chromosomes or sister chromatids (X = 2N cells that split into 1N) to separate normally during nuclear division Ł JH Taylor’s experiment: Sister chromosomes can exchange homologous segments of DNA Down Syndrome (trisomy 21): 3 copies of chrom. # 21 • Autosome: Any chrom. that is not a sex chrom. Mendel’s Laws: 1. Law of Segregation : Two alleles of a gene separate from each other = gametes; half of one allele & other, sister chromosomes held together by cohesin at its kinetochore is separated there. 2. Law of Independent Assortment : Different genes separate independently. R: two diff genes • Meiosis: Part of Gametogenesis (= Gamete formation), providing diff combos everytime Stages of meiosis: 1. Interphase; replication of DNA 2. Prophase I: Recombination, crossing 3. Metaphase I: Line up in middle 4. Anaphase I: First division, homologs 4N > 2*2N PRIMARY N.D. 5. Telophase I: Cytokinesis. 6. Prophase II / Metaphase II 7. Anaphase II: Sister chromatids disjoin, 2* 1N SECONDARY N.D. 8. Telophase II 4 haploid cells • Spermatogenesis: Spermatogonia: Creation of identical mitoses= diploid cells Which grow & mature into a primary spermatocyte, via meiosis = 2 2nd spermatocytes
Meiosis con’t creating 4 haploid spermatids, which differentiate into sperm cells. • Oogenesis: Oogonia: Mitosis creates identical diploid mitoses which grow and differentiate into a primary oocyte > 2nd oocyte with a polar body After fertilization the Oocyte becomes an egg. Lecture 2: • Absorption spectrum: A plot of intensity of light absorbed relative to its wavelength DNA absorbs light in UV range, max. absorption = 260nm, used to determine DNA concentration doublestranded (ds)DNA: • Denaturation: Separation of double helix into two strands When denatured, the absorbance increases 1.5X, because dsDNA cannot move freely while ssDNA can rotate to get max. absorbance; DNA must be positioned at precise location and angle for max. absorption. • Complexity: Measure of # of unique (vs repetitive sequences in a genome), measured by Co t½ • Hybridization: 2 complementary DNA, form together = dsDNA • Renaturing/ Reannealing: Reassociating two single stranded genetic material, via spectrometry Two strands cannot get together, if at higher temp than m.p. Procedure: 1) Purify DNA, to cut to avg. size of 10002000bp 2) Denature then allow to renature at lower temp 3) Measure % renatured by absorption Time for half of molecules to renature = Co t½, Co = [ ] Cot curves: Low [ ] = unique = harder to find hybrid match • In situ hybridization: Finding the original gene location Procedure: 1) Denature, with hot salt solution 2) Incubate in a biotinylated probe (testtube) & wash unhybridized DNA 3) dsDNA hybrid, labeled with fluorescein called avidin to reveal location of bound labeled DNA probe 4) Counterstain DNA to see chromosome • Centromere: the point on a chromosome by which it is attached to a spindle fiber during cell division • Telomere: structure at the end of a chromosome More than one DNA probe glowing = repetitive, many greens fluorescent probe is merged with DNA located the end of a DNA = location of the Telomere Evolution of the Genome: 1) Mutation: the changing of the structure of a gene, resulting in a variant form that may be transmitted to subsequent generations, caused by the alteration of single base units in DNA, or the deletion, insertion, or rearrangement of larger sections of genes or chromosomes. Slow, single base change, selection stabilizes mutation 2) Unequal crossing over: gene duplication or deletion event that deletes a sequence in one strand and replaces
it with a duplication from its homologous chromosome during meiosis Leads to duplications/ deletions. Diverge from mutation/ selection, selection stabilized • Duplication: Arise from unequal crossingover that occurs during meiosis between misaligned homologous chromosomes, creating new genetic material generated • Deletion: a mutation (aberration) in which a part of a chromosome or a sequence of DNA is missing. Deletion is the loss of genetic material; often lethal Ł Evolution of Globins Genes; carrier of O2: 3 Exon fusion & 2 introns, Duplication (2 with extra globin gene), Divergence bc of mutation, separation of genes, with fake gene remains (pseudogenes) • Gene Family: A set of several similar genes, formed by duplication of a single original gene DNA is doubled stranded, anti parallel helixed and a polynucleotide chain, where complementary base (equal amount of each) pairing via Hbonds: AT (2 Hbonds) & GC (3 HBonds) 3.5 picograms (1012) = 3.2 billion base pairs 22 autosomes & XY chromosomes, haploid size = 3 * 10 9 bp 25,000 genes of avg. size 1.5kbp, 1.5% is protein encoding while rest of the genome is repetitive. Lecture 3: • Karyotype: an organized profile of a person's chromosomes • Inversion: A chromosomal aberration when a chromosome is broken in two places, the centre segment is then incorporated in chromosome in reverse order, at the ends of a transposon • Transposition: Ability of genes to change position on chromosomes, a transposable element is removed from one site and inserted into a second site on chromosome = DNA instability, affect gene expression Ł McClintock Corn, Nobel 1983 C Gene controlling maize kernel colour, purple is normal If transposon (= disrupts gene function) jumps out early in kernel development then kernels are mostly purple, if late then colorless kernels with small purple parts (most cell division already occurs) • Transposon: DNA segment capable of moving from one place to another in genome • Genome: Complete set of genes/ genetic material in cell/ organism • Retrotransposon: Transposable elements req. reverse transcriptase for movement in genome • Reverse transcriptase: An RNA dependent DNA polymerase, Using RNA as a template to synthesize complementary strand of DNA Generation of a Direct Repeat: 1) Target site, staggered cuts are made by transposase 2) Leaving end structures the middle is where transposon is inserted 3) Repair synthesis where direct repeats are inserted Method of bacterial transposition (Non replicative): 1) Tansposon DNA between two donor DNA
2) 3) 4) 5)
Transposase binding on ends of transposon Transposon is cleaved from donor Target DNA is placed with cleaved piece Transposon is integrated into the Target DNA The donor DNA rejoins ends after loss of transposon Bacterial transposition (Replicative): 1) Donor with retrotransposon 2) Transcription by RNA polymerase 3) Reverse transcription to single stranded cDNA 4) Conversion to double stranded DNA to insert into target DNA Results in a recipient & donor DNA w/ retrotransposon • Centimorgan: a map unit used to express the distance btwn two gene loci on a chromosome. A spacing of one centimorgan = 1% chance that two genes will be separated by crossing over. • Genetic map: Genetic markers to relative positions on a chromosome based on crossover freq. Examine recombination frequency between two traits (usually visible) to estimate the distance between two genes on the same chromosome (linked genes) If genes are far apart = more crossover recombinants If genes are close together, the recombo freq is lower • Molecular/Physical map: An estimate of the true distance, in base pairs, between 2 items of interest, more precise exact distant btwn genes can be determined Molecular approach uses info from genetic map, to find general order and linkage of genes; use of bioinformatics Both genetic and physical maps provide the likely order of items along a chromosome • Polymorphism: Genetic variation within a population = natural selection operate. The occurrence of something in several different forms There are conserved sequences found in all species, these conserved areas are likely same in all species bc variation in these area will lead to unfunctional regions Ł Speech Gene, FOXP2: Gene that influences fine motor control of lips/tongue, speech impediments in humans are a result of a mutation on FOXP2. Apes have a substitution in his region • Restriction fragment length polymorphism (RFLP): a base change resulting in abolition/ creation of a recognition sequence for a restriction enzyme (= R. enzymes in bacteria will recognize sequence & cleave DNA at specific recog. sites (100x exists) Eg. EcoR1 digest, a mutation in the EcoRI recognition sites creates an RFLP, only works well on sites that has that sequence, if not recognize less variation on fragment size • Single nucleotide polymorphism (SNP): a base change, which may or may not lead to a RFLP. Many must be detected by direct DNA sequencing. Millions of SNPs in human genome, Individuals differ by 3mil SNPs, correlate SNP pattern with disease, drug reaction Lecture 4: Ł Beadle and Tatum Experiment Proposal of One geneone enzyme hypothesis: Production of a protein is encoded by a gene and enzymes are produced from single or group of protein(s)
1) Initially a wildtype neurospora as able to be grown in minimal medium to create mutations spores were exposed to radiation 2) Neurospora was then grown in supplemented medium and following meiosis the mutated spores were taken to see if they could grow in minimal medium; they couldn’t only in supplemented medium 3) Mutants now tested in two different minimal mediums: one supplemented with amino acids the other sup. with vitamins, only vitamin supplemented produced growth; this resulted in observation that mutant had deficiency in enzyme to form vitamins (biosynthesis) hence needed vitamins. 4) Further tested with several vitamins = mutation in gene encoding protein involved in making the vitamin pantothenic acid. P. acid is part of making Coenzyme A, if provide with P. acid, it can bypass the mutant block = norm growth • Transcription: The process by which genetic information (DNA) nucleotides = synthesized molecules of RNA, with the DNA serving as a template. Transcribed in nucleus = pre mRNA, processed into mRNA mRNA transported out to cytoplasm to be translated by ribosomes into polypetides, the folded = mature Prokaryotic RNA polymerase consists of several subunits & a one type of polymerase for all transcriptional activities. 1) RNA polymerase transcribes DNA into RNA, from 5’ to 3’ 2) Unwinding of DNA = arrangement stress; alleviated by supercoiling; NTP hydrolysis provides energy 3) Many polymerases can act on same gene at the same time • Core enzyme: A single type of RNA Polymerase composed of 5 subunits tightly associated If core enzyme is purified from bacterial cells (eg E. Coli) and added to bacterial DNA, enzyme binds to DNA and synthesizes RNA at random initiation sites • Sigma factor: A purified accessory polypeptide that is added to RNA polymerase before attachment to DNA, transcription will begin at select locations Sigma factor binds to sequences at 10/35, when sigma factor is lost = RNA chain is elongated • Holoenzyme: Sigma factor + core enzyme = complete & = proper opening of double helix • TATA box: Regulatory sequence that is most proximal upstream (from start site) and also a core promoter element (= site of assembly of preinitiation complex of RNA Poly II & general transcription factors before transcription occurs) A promoter is a region upstream (away from start site) from agene that regulates initiation of transcription Eg. PEPCK gene (phosphoenolpyruvate carboxykinse); lacking TATA box = less transcription; or downstream promoter element, down stream from start site • Consensus sequence: The most common version of a conserved sequence (= amino acid sequences of particular polypeptide/ nucleotide, homologous = haven’t diverged = conserved over evolution) exist upstream (5’ to…) the gene they control and RNA polymerase interacts and specifically binds to some of these. found at 35 and 10 in prokaryotes
Most eukaryotic RNAs are synthesized as preRNAs, which are then processed to yield a functional mature RNA • Ribosome: protein complex, found in large numbers in the cytoplasm of living cells. They bind messenger RNA and transfer RNA to synthesize polypeptides and proteins. • Nucleolus: Incomplete rRNA genes at the site of ribosome assembly (cytoplasm) • Nucleolar organizer region (NOR): Where ribosomal RNA (rRNA = RNA of a ribosome) is synthesized (specific sites within the nucleus) Contains many tandemly repeated (=cluster of DNA sequence that repeats itself without interruption) copies of the rRNA genes. 1. RNA Polymerase: an enzyme that brings about the formation of a polymer RNA; multisubunit complex. In eukaryotes, three different polymerases are used and are separated by their receptiveness to αamanitin: 2. Pol I: responsible for synthesis of large (rRNAs) 3. Pol II: mRNAs 4. Pol III: small RNAs (e.g. tRNA). Penman Experiment: 1) Pulse and Chase Pulse chase experiments were active in deciphering how processing occurs; a basic biochemical tool; molecule or pop. of molecules = determine fate over a time course. Pulse: add radioactive precursor to cells for short period. Merging of label into macromolecule pool Chase: Wash “chase away” the radioactive precursor then examine the radioactive macromolecules after an incubation period Plotting fraction # against another parameter (absorbance value eg. optical density): highest [ ] of fractions is middle sized, but three distinct peaks 2) Nucleolar and cytoplasmic fractions were prepared 3) RNA Isolated 4) Separate RNA by size, (centrifuge) a. Svedberg (S Value): Sedimentation coefficient of RNA; the S value is correlated with size and shape, so S values are generally not additive. Sedimentation/Centrifugation: 1) In sucrose solution, spun in centrifuge 2) Small at the top, middle and large at bottom. 3) Poke hole, drip from largest to smallest, collect fractions Primary rRNA transcripts = 45S, processes to 28, 18 & 5.8S 5) Determine the distribution of RNA & follow radio labeled RNA RNA processing: 45S 32S 41S 28S 18S 5.8S 28S 18S 5.8S 5.8S, 18S and 28S are stable rRNA that assemble with proteins and build ribosomes The RNA absorbance (= measure of amount of RNA of each class) remains constant from 10 mins to 150mins, in nucleolus
with mostly 45S & 32S. As for absorbance in cytoplasm, remains similar of 28 > 18 > 4S The radioactivity increases from 10mins to 150min from most activity in the 45S then 32S in nucleolus. Next, the radioactivity in cytoplasm mostly in 4S then all of 18 & 28S has activity by 150mins Lecture 5: • Heterogeneous nuclear RNA (hnRNA): Large group of RNA molecules (incld. Pre mRNA) that have large molecular weights (up to 80S), represent many different nucleotide sequences & only found in nucleus 1. Pulse and chase method used to observe the conversion of hnRNA to smaller mRNA a. Label cells for short period with 32p, purifed RNA, centrifuge to separate molecules by size, determine optimal density & radioactivity = newly synthesized RNA are very large b. Chased, and found that large small because no presence of hnRNA, 90% rRNA because rRNA is most stable (half life of hnRNA is min – hrs while rRNA is day – months = accumulation of them) Eukary. mRNAs are created from these larger precursors Expression Control 1. Nuclear Gene is transcribed by RNA polymerase II (which binds upstream 5’ to 3’ of the gene to be transcribed, addition or loss of subunits, phosphorylation of subunits alter its activity such that initiation, elongation and termination) Transcription of a poly II gene is regulated by transcription factors that recruit RNA polymerase & sequences in the promoter of the gene (binding sites for poly II subunits) 2. hnRNA is processed into mRNA 3. Exported to the cytoplasm 4. Translation Initiation of transcription from an eukaryotic polymerase II promoter 1) TATA binding protein (RBP) bends DNA, partial unwinding occurs to serve as binding site. 2) Other transcription factors recruited by TBP & TBP associated factors (TAF) • RNA polymerase is held in place until TFIIH (= a Protein kinase: Enzyme that transfers phosphate groups to other proteins = often regulates activities of other proteins = switch) phosphoryates the carboxyl terminal domain of polymerase II where 52 repeats of 7 amino acids exists • Preinitiation complex: The state where all the factors are recruited and the nonphosphoryated tail, once the tail is phosphorylated = tail released • Phosphorylation: Introduce a phosphate group into (a molecule or compound). • mRNA cap: The 5’ methylguanosine cap before the 5’ untranslated region, nucleases work from 5’ to 3’, this cap prevents the 5’ end to be digested by nucleases, aids in transport of mRNA, role in initiation of mRNA translation
Found @ 7th position of Guanosine (= consisting of guanine combined with ribose) is methylated as are 2’ positions of riboses 1 & 2 (= a sugar of the pentose class, a constituent of nucleosides and enzymes) • Poly A tail: Initially, begins as a 20 nucleotides sequence that serves as recognition site for proteins; associated with RNA poly II. A string of adenosine residue at the 3’ end of a mRNA added after transcription, length variable 250 nucleotides, is to protect mRNA from premature degradation by exonucleases • Untranslated region (UTRs): Noncoding segments contained at both 5’ and 3’ ends of mRNA A mature mRNA usually consists of a 5’ cap, the 5’ untranslated leader, the ‘coding’ region, the 3’ untranslated region, and a poly A tail. • Complementary DNA (cDNA): Relationship between sequences of bases in the two strands of double helix of DNA, nucleotides adeninethymine and guanine cytosine; LACKS INTRONS • Reverse transcriptase*
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Genomic clone: Contains both introns and exons
Exon: Parts of a split gene that contribute to a mature RNA product; containing information coding for a protein or peptide sequence • Intron: Part of a split gene (= genes with intervening sequences), a segment of a DNA or RNA molecule that does not code for proteins and interrupts the sequence of genes 1) After hnRNA has 5’ capping and poly A tail added 2) Removal of 3’ end by nuclease and polyadenylation Removal of introns occurs by RNA splicing reactions. Splicing can be autonomous (selfsplicing RNAs), or mediated by a multisubunit complex = spliceosome. 3) Splice junction: Endonucleolytic cleavage of intron at these locations 4) Ligation of exons; exons remove self and attach back together By comparing the DNA sequences of genomic and cDNA clones, conserved splice junction sequences were discovered; Consensus sequences at the intron/exon junctions are used to identify the proper splice sites. Hybridization assays and DNA sequencing are useful for determining exactly what has occurred in mRNA maturation. Lecture 6 • Consensus sequence:*
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Frame shift mutation/Missense mutation: Mutations which a single base pair is either added or deleted from DNA , this results in incorrect reading frame from point of mutation (everything is shifted) • Nonsense mutation: Mutations that product stop codons within genes causing premature termination of the encoded polypeptide chain 1982; Cech discovers selfsplicing RNA in the Tetrahymena Incorrect splicing = mutations; protein function is stopped ie. not exported to cytoplasm, degraded & no protein prod.
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Self Splicing Group 1 introns: found in nuclear RNA of lower eukaryotes, some organelles forms elaborate 3D structure due to intrastrand basepairing • Self Splicing Group 2 introns: Discovered in fungal mitochondria, These introns undergo self splicing; found in organelles • Lariat: Intermediate of group 2 introns, shape that resembles a loop, with two exons lined up. Self splicing in group 2 introns 1. The A site at the 2’ attaches to the G site at the 5’ end creating a loop shape = phosphodiester bond 2. The free 3’ OH of displaced exon attacks the 3’ splice site on beginning of exon 2, intron is then released as a free lariat Northern blotting (RNA gel blotting) : used to demonstrate of RNA splicing, separation of RNA on gel, hybridization with specific gene probe; Can be used to show that RNA processing occurs stepwise, showing the origin and molecular weight of the fragents, low = complete RNA 1. A pop. of RNA; electrophoresis to separate by size 2. Blot onto a sheet of paper; solid support; immobilized RNA 3. Add gene probe where only complimentary RNA will bind 4. Wash and expose to xray film to detect radio labeled hybrids Spliceosome: In higher eukaryotes, Macromolecular complex containing various proteins and ribonucleoproteins that functions to remove introns from primary transcript • snRNP: AKA “snurps” Specific ribonucleoprotein particles contained in splicosomes, composed of snRNAs bound to specific proteins • Small Nuclear RNAs (snRNAs): RNAs req for mRNA processing, function in nucleus called U1,U2…U6 Base pairing between snRNAs and between snRNAs and hnRNA intron sequences strategically position molecules for cleavage and religation events Spliceosome mediated RNA splicing: 1. U1 binds to splice donor 2. U2 binds to branch point (the A site); both base pair w/ conserved sequences 3. U4/U6 interstrand pairing; A lariat is formed by the intron 4. U6 displaces U1, acts as ribozyme; Cleavage occurs at the splice donor site. 5. Extensive pairing btwn snRNAs & snRNA/hnRNA, The adjacent exons are ligated together ultimately makes A site closer to splicing site Splicing/polyadenylation factors associate with phosphorylated CTD (Carbon terminal domain) of RNA polymerase; organizes factors for capping, intron removal • Ribozyme: An RNA molecule that functions as a catalyst in cellular reactions; Selfsplicing implies that RNA has catalytic activity, like an enzyme New riboenzymes can be created by synthesizing random chains of RNA, but their functionality is a question; used of an assay (= testing of catalytic activity)
Ability to bind to amino acid lysine and tRNA means that a riboenzyme identified have function equivalent of aminoacyl tRNA synthetases. Riboenzymes used in AIDs treatment by stopping the ADIS mRNA by binding AIDS mRNA & cleaving it or creation of AIDS proteins stopped by riboenzyme binding to AIDs protein & cleaving it. • Small interfering RNA (siRNA): 21 23 nucleotide, double stranded fragments formed when double stranded RNA initiates DNA silencing • RNA Silencing: A process where small noncoding RNA (derived from longer dsPrecursors) trigger sequence specific inhibition of gene expression Jorgensen flower pigment: Light purple flower expected to be more purple when added more purple pigments but observed is an albino flower, both endogenous and added genes were expressed but RNA encoded were destroyed = post transcriptional gene silencing. • Micro RNA (miRNA): Small RNA 2023 nu, synthesized from many sites in the genome and involved in inhibiting translation or increasing degradation of complementary mRNA Small interfering RNAs (siRNAs) and microRNAs (miRNAs) play roles in development and defense by mediating destruction of dsRNA or inhibiting translation Formation & mechanism of action siRNA miRNA; bobby pin 1 Origin: dsDNA is Origin: ssprecursor RNA cleaved by that contains complementary endonucleases dicer to sequences that allow them to small siRNA, over fold into pseudo dsRNA (pri hanging ends miRNA), cleaved by endonuclease Drosha at terminal c==* VAL Ł To elucidating the genetic code: 1) A new tool; In vitro translation system 1. Lyse cells = centrifuge to remove membranes 2. Addition of radioactive amino acids with RNA = radio active protein [ ] ^ 2) An organic chemist; Org. synthesis of polynucleotides 1. Bacterial extract with synthetic mRNA makes polypeptides, = 3/64 triplets 3) Brute force 1. Organic synthesis cannot control the ratios of nucleotides made, the relative [ ] of triplets is statistically determined, analyzed amino acid content of the protein produced by this mixture Random copolymer UUUG Eg. Codon: p(UUU) = 3*(¾) = 27/64 of getting UUU 4) Brilliance 1) Kgorana discovered how to synthesize defined triplets 2) Nirenberg & Ledger: filter binding assays I. Set up 20 cell free reactions each with 1 radioactive amino acid and 19 unlabelled amino acids II. Give each with same defined triplet III. Filter, to determine which is radioactive IV. Bound amino acid + trinucleotide retained in filter, in all other cases the AA* will go through All triplets decoded this way • Codon: Sequence of a nucleotides triplet in mRNA that specify amino acids the ‘genetic code’ is a triplet code, with each triplet set of nucleotides in RNA decoded into an amino acid. Three of the 64 codons are ‘stop’ codons that signal the end of the polypeptide chain. • Anticodon: A three nucleotide sequence in each tRNA that functions in recognition of the complementary mRNA codon • Transfer RNA (tRNA): small RNAs (~75AA) that translates the info encoded in nucleotide alphabet of an mRNA into amino acid alphabet of a polypeptide, tRNAs = adaptors. The anticodon of a tRNA forms complementary base pairs with the triplet codon in mRNA. tRNA brings proper amino acid to ribosome The anticodon site for amino acid attachement is at the 5’ end on the amino acid acceptor stem • Wobble hypothesis: Crick’s proposal that the steric req between the anticodon of the tRNA and the codon of
the mRNA is flexible at the 3rd position = only allow two codons that differ at 3rd to share the same tRNA during protein synthesis Often the 5’ nucleotide of tRNA anticodon will base pari with any 3’ nucleotide of the codon = same tRNA > 1codon Wobble base: Codon/Anti codon Anticodon Codon (wobblebase) U A or G G U or C I inosine derived from G U, C or A • Amino acyl tRNA synthetase (AARS): An enzyme that recognizes and covalently link amino acids to the 3’ ends of their equivalent tRNA, each amino acid is recognize by specific AARS, Catalyze two step reaction joining AA + tRNA Amino acid activation: ATP + AA AAAMP + PPi tRNA charging: AAAMP + tRNA AAtRNA + AMP Mechanism for translation: 1. Initiation codon: AUG, the site where ribosome attaches to the mRNA at assure that the ribosome is in the proper reading frame to reach entire msg or a missence protein is produced. In Eukaryotes Initiator tRNA finds mRNA 5’ cap structure recognized by initiation factor & binds, multicomplex formed and scans til identifies start codon, consensus sequence around AUG 2. Elongation: Cycles of elongation add amino acids to the growing chain, ribosome must move relative to mRNA Tu are elongation factors that bring AAtRNA to ribosome when bound to GTP = TGDP Ts are elongation factors that sponsors exchange of GTP for GDP on Tu = reactivates Tu TuGTP + AAtRNA AAtRNA to ribosome Process of elongation APE sites: a) At site A peptide bond formation by Peptidyl transferase: [Portion of the large ribosomal subunit that is responsible for catalyzing peptide bond formation] Peptide bond formation when (Psite)OH + H (Asite) H2O (water is released) to create single bond between amino & carboxy group b) Translocation: [move (a portion of a chromosome) to a new position on the same or another chromosome] occurs moving peptidyl tRNA to P site, GTP req c) A Site is empty, ready for next codon decoding d) Release of uncharged tRNA from E Site 3. Termination: Decoding of stop codon (3) as a termination signal, release of mature polypeptide No tRNA to decode = stop Release factors bind, GTP hydrolysis Complete polypeptides are released Ribosomes are dissociates from mRNA Overall for translation need: mRNA (template for codon), functional ribosome (decoding, moving), amino acyltRNA (Adapt and read codon, bring AA), energy (driving) In prokaryotes, a ShineDalgarno sequence 5’ to the AUG start codon in mRNA 1. Small ribosome subunit & initiation factors req, GTP required, tRNA 2. A,P,E sites: The ribosome has 3 functional sites
A = Aminoacyl/ Acceptor P = Peptidyl E = Exit
Incoming AAtRNA bind Growing polypeptide is held in place Uncharged tRNAs are released
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Polysome (polyribosome): The complex formed by an mRNA & a number of ribosomes in the process of translating that mRNA Implicates that many copies of a protein can be produced from a single mRNA
Lecture 9: Nuclear & chromatin structure
• Dominant/ Recessive Genes: Homozygous dominant aka wildtype • Homologous chromosome: Paired chromosomes of diploid cells, that are similar in structure; contains two sister chromatids • Bivalent/Tetrad: Formed during meiosis by junction of two homologous pair • Recombination/ Reciprocal genetic exchange: Genetic rearrangement of genetic material via crossing over or formation of bivalent • Homozygous: two identical alleles of a gene • •
Heterozygous: two different alleles of a gene
Synaptonemal complex: a ladderlike series of parallel threads holding pairing homologous chromosomes in meiosis for recombination to occur. • Nondisjunction: the failure of one or more pairs of homologous chromosomes or sister chromatids (X = 2N cells that split into 1N) to separate normally during nuclear division Ł JH Taylor’s experiment: Sister chromosomes can exchange homologous segments of DNA Down Syndrome (trisomy 21): 3 copies of chrom. # 21 • Autosome: Any chrom. that is not a sex chrom. Mendel’s Laws: 1. Law of Segregation : Two alleles of a gene separate from each other = gametes; half of one allele & other, sister chromosomes held together by cohesin at its kinetochore is separated there. 2. Law of Independent Assortment : Different genes separate independently. R: two diff genes • Meiosis: Part of Gametogenesis (= Gamete formation), providing diff combos everytime Stages of meiosis: 1. Interphase; replication of DNA 2. Prophase I: Recombination, crossing 3. Metaphase I: Line up in middle 4. Anaphase I: First division, homologs 4N > 2*2N PRIMARY N.D. 5. Telophase I: Cytokinesis. 6. Prophase II / Metaphase II 7. Anaphase II: Sister chromatids disjoin, 2* 1N SECONDARY N.D. 8. Telophase II 4 haploid cells • Spermatogenesis: Spermatogonia: Creation of identical mitoses= diploid cells Which grow & mature into a primary spermatocyte, via meiosis = 2 2nd spermatocytes
Meiosis con’t creating 4 haploid spermatids, which differentiate into sperm cells. • Oogenesis: Oogonia: Mitosis creates identical diploid mitoses which grow and differentiate into a primary oocyte > 2nd oocyte with a polar body After fertilization the Oocyte becomes an egg. Lecture 2: • Absorption spectrum: A plot of intensity of light absorbed relative to its wavelength DNA absorbs light in UV range, max. absorption = 260nm, used to determine DNA concentration doublestranded (ds)DNA: • Denaturation: Separation of double helix into two strands When denatured, the absorbance increases 1.5X, because dsDNA cannot move freely while ssDNA can rotate to get max. absorbance; DNA must be positioned at precise location and angle for max. absorption. • Complexity: Measure of # of unique (vs repetitive sequences in a genome), measured by Co t½ • Hybridization: 2 complementary DNA, form together = dsDNA • Renaturing/ Reannealing: Reassociating two single stranded genetic material, via spectrometry Two strands cannot get together, if at higher temp than m.p. Procedure: 1) Purify DNA, to cut to avg. size of 10002000bp 2) Denature then allow to renature at lower temp 3) Measure % renatured by absorption Time for half of molecules to renature = Co t½, Co = [ ] Cot curves: Low [ ] = unique = harder to find hybrid match • In situ hybridization: Finding the original gene location Procedure: 1) Denature, with hot salt solution 2) Incubate in a biotinylated probe (testtube) & wash unhybridized DNA 3) dsDNA hybrid, labeled with fluorescein called avidin to reveal location of bound labeled DNA probe 4) Counterstain DNA to see chromosome • Centromere: the point on a chromosome by which it is attached to a spindle fiber during cell division • Telomere: structure at the end of a chromosome More than one DNA probe glowing = repetitive, many greens fluorescent probe is merged with DNA located the end of a DNA = location of the Telomere Evolution of the Genome: 1) Mutation: the changing of the structure of a gene, resulting in a variant form that may be transmitted to subsequent generations, caused by the alteration of single base units in DNA, or the deletion, insertion, or rearrangement of larger sections of genes or chromosomes. Slow, single base change, selection stabilizes mutation 2) Unequal crossing over: gene duplication or deletion event that deletes a sequence in one strand and replaces
it with a duplication from its homologous chromosome during meiosis Leads to duplications/ deletions. Diverge from mutation/ selection, selection stabilized • Duplication: Arise from unequal crossingover that occurs during meiosis between misaligned homologous chromosomes, creating new genetic material generated • Deletion: a mutation (aberration) in which a part of a chromosome or a sequence of DNA is missing. Deletion is the loss of genetic material; often lethal Ł Evolution of Globins Genes; carrier of O2: 3 Exon fusion & 2 introns, Duplication (2 with extra globin gene), Divergence bc of mutation, separation of genes, with fake gene remains (pseudogenes) • Gene Family: A set of several similar genes, formed by duplication of a single original gene DNA is doubled stranded, anti parallel helixed and a polynucleotide chain, where complementary base (equal amount of each) pairing via Hbonds: AT (2 Hbonds) & GC (3 HBonds) 3.5 picograms (1012) = 3.2 billion base pairs 22 autosomes & XY chromosomes, haploid size = 3 * 10 9 bp 25,000 genes of avg. size 1.5kbp, 1.5% is protein encoding while rest of the genome is repetitive. Lecture 3: • Karyotype: an organized profile of a person's chromosomes • Inversion: A chromosomal aberration when a chromosome is broken in two places, the centre segment is then incorporated in chromosome in reverse order, at the ends of a transposon • Transposition: Ability of genes to change position on chromosomes, a transposable element is removed from one site and inserted into a second site on chromosome = DNA instability, affect gene expression Ł McClintock Corn, Nobel 1983 C Gene controlling maize kernel colour, purple is normal If transposon (= disrupts gene function) jumps out early in kernel development then kernels are mostly purple, if late then colorless kernels with small purple parts (most cell division already occurs) • Transposon: DNA segment capable of moving from one place to another in genome • Genome: Complete set of genes/ genetic material in cell/ organism • Retrotransposon: Transposable elements req. reverse transcriptase for movement in genome • Reverse transcriptase: An RNA dependent DNA polymerase, Using RNA as a template to synthesize complementary strand of DNA Generation of a Direct Repeat: 1) Target site, staggered cuts are made by transposase 2) Leaving end structures the middle is where transposon is inserted 3) Repair synthesis where direct repeats are inserted Method of bacterial transposition (Non replicative): 1) Tansposon DNA between two donor DNA
2) 3) 4) 5)
Transposase binding on ends of transposon Transposon is cleaved from donor Target DNA is placed with cleaved piece Transposon is integrated into the Target DNA The donor DNA rejoins ends after loss of transposon Bacterial transposition (Replicative): 1) Donor with retrotransposon 2) Transcription by RNA polymerase 3) Reverse transcription to single stranded cDNA 4) Conversion to double stranded DNA to insert into target DNA Results in a recipient & donor DNA w/ retrotransposon • Centimorgan: a map unit used to express the distance btwn two gene loci on a chromosome. A spacing of one centimorgan = 1% chance that two genes will be separated by crossing over. • Genetic map: Genetic markers to relative positions on a chromosome based on crossover freq. Examine recombination frequency between two traits (usually visible) to estimate the distance between two genes on the same chromosome (linked genes) If genes are far apart = more crossover recombinants If genes are close together, the recombo freq is lower • Molecular/Physical map: An estimate of the true distance, in base pairs, between 2 items of interest, more precise exact distant btwn genes can be determined Molecular approach uses info from genetic map, to find general order and linkage of genes; use of bioinformatics Both genetic and physical maps provide the likely order of items along a chromosome • Polymorphism: Genetic variation within a population = natural selection operate. The occurrence of something in several different forms There are conserved sequences found in all species, these conserved areas are likely same in all species bc variation in these area will lead to unfunctional regions Ł Speech Gene, FOXP2: Gene that influences fine motor control of lips/tongue, speech impediments in humans are a result of a mutation on FOXP2. Apes have a substitution in his region • Restriction fragment length polymorphism (RFLP): a base change resulting in abolition/ creation of a recognition sequence for a restriction enzyme (= R. enzymes in bacteria will recognize sequence & cleave DNA at specific recog. sites (100x exists) Eg. EcoR1 digest, a mutation in the EcoRI recognition sites creates an RFLP, only works well on sites that has that sequence, if not recognize less variation on fragment size • Single nucleotide polymorphism (SNP): a base change, which may or may not lead to a RFLP. Many must be detected by direct DNA sequencing. Millions of SNPs in human genome, Individuals differ by 3mil SNPs, correlate SNP pattern with disease, drug reaction Lecture 4: Ł Beadle and Tatum Experiment Proposal of One geneone enzyme hypothesis: Production of a protein is encoded by a gene and enzymes are produced from single or group of protein(s)
1) Initially a wildtype neurospora as able to be grown in minimal medium to create mutations spores were exposed to radiation 2) Neurospora was then grown in supplemented medium and following meiosis the mutated spores were taken to see if they could grow in minimal medium; they couldn’t only in supplemented medium 3) Mutants now tested in two different minimal mediums: one supplemented with amino acids the other sup. with vitamins, only vitamin supplemented produced growth; this resulted in observation that mutant had deficiency in enzyme to form vitamins (biosynthesis) hence needed vitamins. 4) Further tested with several vitamins = mutation in gene encoding protein involved in making the vitamin pantothenic acid. P. acid is part of making Coenzyme A, if provide with P. acid, it can bypass the mutant block = norm growth • Transcription: The process by which genetic information (DNA) nucleotides = synthesized molecules of RNA, with the DNA serving as a template. Transcribed in nucleus = pre mRNA, processed into mRNA mRNA transported out to cytoplasm to be translated by ribosomes into polypetides, the folded = mature Prokaryotic RNA polymerase consists of several subunits & a one type of polymerase for all transcriptional activities. 1) RNA polymerase transcribes DNA into RNA, from 5’ to 3’ 2) Unwinding of DNA = arrangement stress; alleviated by supercoiling; NTP hydrolysis provides energy 3) Many polymerases can act on same gene at the same time • Core enzyme: A single type of RNA Polymerase composed of 5 subunits tightly associated If core enzyme is purified from bacterial cells (eg E. Coli) and added to bacterial DNA, enzyme binds to DNA and synthesizes RNA at random initiation sites • Sigma factor: A purified accessory polypeptide that is added to RNA polymerase before attachment to DNA, transcription will begin at select locations Sigma factor binds to sequences at 10/35, when sigma factor is lost = RNA chain is elongated • Holoenzyme: Sigma factor + core enzyme = complete & = proper opening of double helix • TATA box: Regulatory sequence that is most proximal upstream (from start site) and also a core promoter element (= site of assembly of preinitiation complex of RNA Poly II & general transcription factors before transcription occurs) A promoter is a region upstream (away from start site) from agene that regulates initiation of transcription Eg. PEPCK gene (phosphoenolpyruvate carboxykinse); lacking TATA box = less transcription; or downstream promoter element, down stream from start site • Consensus sequence: The most common version of a conserved sequence (= amino acid sequences of particular polypeptide/ nucleotide, homologous = haven’t diverged = conserved over evolution) exist upstream (5’ to…) the gene they control and RNA polymerase interacts and specifically binds to some of these. found at 35 and 10 in prokaryotes
Most eukaryotic RNAs are synthesized as preRNAs, which are then processed to yield a functional mature RNA • Ribosome: protein complex, found in large numbers in the cytoplasm of living cells. They bind messenger RNA and transfer RNA to synthesize polypeptides and proteins. • Nucleolus: Incomplete rRNA genes at the site of ribosome assembly (cytoplasm) • Nucleolar organizer region (NOR): Where ribosomal RNA (rRNA = RNA of a ribosome) is synthesized (specific sites within the nucleus) Contains many tandemly repeated (=cluster of DNA sequence that repeats itself without interruption) copies of the rRNA genes. 1. RNA Polymerase: an enzyme that brings about the formation of a polymer RNA; multisubunit complex. In eukaryotes, three different polymerases are used and are separated by their receptiveness to αamanitin: 2. Pol I: responsible for synthesis of large (rRNAs) 3. Pol II: mRNAs 4. Pol III: small RNAs (e.g. tRNA). Penman Experiment: 1) Pulse and Chase Pulse chase experiments were active in deciphering how processing occurs; a basic biochemical tool; molecule or pop. of molecules = determine fate over a time course. Pulse: add radioactive precursor to cells for short period. Merging of label into macromolecule pool Chase: Wash “chase away” the radioactive precursor then examine the radioactive macromolecules after an incubation period Plotting fraction # against another parameter (absorbance value eg. optical density): highest [ ] of fractions is middle sized, but three distinct peaks 2) Nucleolar and cytoplasmic fractions were prepared 3) RNA Isolated 4) Separate RNA by size, (centrifuge) a. Svedberg (S Value): Sedimentation coefficient of RNA; the S value is correlated with size and shape, so S values are generally not additive. Sedimentation/Centrifugation: 1) In sucrose solution, spun in centrifuge 2) Small at the top, middle and large at bottom. 3) Poke hole, drip from largest to smallest, collect fractions Primary rRNA transcripts = 45S, processes to 28, 18 & 5.8S 5) Determine the distribution of RNA & follow radio labeled RNA RNA processing: 45S 32S 41S 28S 18S 5.8S 28S 18S 5.8S 5.8S, 18S and 28S are stable rRNA that assemble with proteins and build ribosomes The RNA absorbance (= measure of amount of RNA of each class) remains constant from 10 mins to 150mins, in nucleolus
with mostly 45S & 32S. As for absorbance in cytoplasm, remains similar of 28 > 18 > 4S The radioactivity increases from 10mins to 150min from most activity in the 45S then 32S in nucleolus. Next, the radioactivity in cytoplasm mostly in 4S then all of 18 & 28S has activity by 150mins Lecture 5: • Heterogeneous nuclear RNA (hnRNA): Large group of RNA molecules (incld. Pre mRNA) that have large molecular weights (up to 80S), represent many different nucleotide sequences & only found in nucleus 1. Pulse and chase method used to observe the conversion of hnRNA to smaller mRNA a. Label cells for short period with 32p, purifed RNA, centrifuge to separate molecules by size, determine optimal density & radioactivity = newly synthesized RNA are very large b. Chased, and found that large small because no presence of hnRNA, 90% rRNA because rRNA is most stable (half life of hnRNA is min – hrs while rRNA is day – months = accumulation of them) Eukary. mRNAs are created from these larger precursors Expression Control 1. Nuclear Gene is transcribed by RNA polymerase II (which binds upstream 5’ to 3’ of the gene to be transcribed, addition or loss of subunits, phosphorylation of subunits alter its activity such that initiation, elongation and termination) Transcription of a poly II gene is regulated by transcription factors that recruit RNA polymerase & sequences in the promoter of the gene (binding sites for poly II subunits) 2. hnRNA is processed into mRNA 3. Exported to the cytoplasm 4. Translation Initiation of transcription from an eukaryotic polymerase II promoter 1) TATA binding protein (RBP) bends DNA, partial unwinding occurs to serve as binding site. 2) Other transcription factors recruited by TBP & TBP associated factors (TAF) • RNA polymerase is held in place until TFIIH (= a Protein kinase: Enzyme that transfers phosphate groups to other proteins = often regulates activities of other proteins = switch) phosphoryates the carboxyl terminal domain of polymerase II where 52 repeats of 7 amino acids exists • Preinitiation complex: The state where all the factors are recruited and the nonphosphoryated tail, once the tail is phosphorylated = tail released • Phosphorylation: Introduce a phosphate group into (a molecule or compound). • mRNA cap: The 5’ methylguanosine cap before the 5’ untranslated region, nucleases work from 5’ to 3’, this cap prevents the 5’ end to be digested by nucleases, aids in transport of mRNA, role in initiation of mRNA translation
Found @ 7th position of Guanosine (= consisting of guanine combined with ribose) is methylated as are 2’ positions of riboses 1 & 2 (= a sugar of the pentose class, a constituent of nucleosides and enzymes) • Poly A tail: Initially, begins as a 20 nucleotides sequence that serves as recognition site for proteins; associated with RNA poly II. A string of adenosine residue at the 3’ end of a mRNA added after transcription, length variable 250 nucleotides, is to protect mRNA from premature degradation by exonucleases • Untranslated region (UTRs): Noncoding segments contained at both 5’ and 3’ ends of mRNA A mature mRNA usually consists of a 5’ cap, the 5’ untranslated leader, the ‘coding’ region, the 3’ untranslated region, and a poly A tail. • Complementary DNA (cDNA): Relationship between sequences of bases in the two strands of double helix of DNA, nucleotides adeninethymine and guanine cytosine; LACKS INTRONS • Reverse transcriptase*
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Genomic clone: Contains both introns and exons
Exon: Parts of a split gene that contribute to a mature RNA product; containing information coding for a protein or peptide sequence • Intron: Part of a split gene (= genes with intervening sequences), a segment of a DNA or RNA molecule that does not code for proteins and interrupts the sequence of genes 1) After hnRNA has 5’ capping and poly A tail added 2) Removal of 3’ end by nuclease and polyadenylation Removal of introns occurs by RNA splicing reactions. Splicing can be autonomous (selfsplicing RNAs), or mediated by a multisubunit complex = spliceosome. 3) Splice junction: Endonucleolytic cleavage of intron at these locations 4) Ligation of exons; exons remove self and attach back together By comparing the DNA sequences of genomic and cDNA clones, conserved splice junction sequences were discovered; Consensus sequences at the intron/exon junctions are used to identify the proper splice sites. Hybridization assays and DNA sequencing are useful for determining exactly what has occurred in mRNA maturation. Lecture 6 • Consensus sequence:*
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Frame shift mutation/Missense mutation: Mutations which a single base pair is either added or deleted from DNA , this results in incorrect reading frame from point of mutation (everything is shifted) • Nonsense mutation: Mutations that product stop codons within genes causing premature termination of the encoded polypeptide chain 1982; Cech discovers selfsplicing RNA in the Tetrahymena Incorrect splicing = mutations; protein function is stopped ie. not exported to cytoplasm, degraded & no protein prod.
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Self Splicing Group 1 introns: found in nuclear RNA of lower eukaryotes, some organelles forms elaborate 3D structure due to intrastrand basepairing • Self Splicing Group 2 introns: Discovered in fungal mitochondria, These introns undergo self splicing; found in organelles • Lariat: Intermediate of group 2 introns, shape that resembles a loop, with two exons lined up. Self splicing in group 2 introns 1. The A site at the 2’ attaches to the G site at the 5’ end creating a loop shape = phosphodiester bond 2. The free 3’ OH of displaced exon attacks the 3’ splice site on beginning of exon 2, intron is then released as a free lariat Northern blotting (RNA gel blotting) : used to demonstrate of RNA splicing, separation of RNA on gel, hybridization with specific gene probe; Can be used to show that RNA processing occurs stepwise, showing the origin and molecular weight of the fragents, low = complete RNA 1. A pop. of RNA; electrophoresis to separate by size 2. Blot onto a sheet of paper; solid support; immobilized RNA 3. Add gene probe where only complimentary RNA will bind 4. Wash and expose to xray film to detect radio labeled hybrids Spliceosome: In higher eukaryotes, Macromolecular complex containing various proteins and ribonucleoproteins that functions to remove introns from primary transcript • snRNP: AKA “snurps” Specific ribonucleoprotein particles contained in splicosomes, composed of snRNAs bound to specific proteins • Small Nuclear RNAs (snRNAs): RNAs req for mRNA processing, function in nucleus called U1,U2…U6 Base pairing between snRNAs and between snRNAs and hnRNA intron sequences strategically position molecules for cleavage and religation events Spliceosome mediated RNA splicing: 1. U1 binds to splice donor 2. U2 binds to branch point (the A site); both base pair w/ conserved sequences 3. U4/U6 interstrand pairing; A lariat is formed by the intron 4. U6 displaces U1, acts as ribozyme; Cleavage occurs at the splice donor site. 5. Extensive pairing btwn snRNAs & snRNA/hnRNA, The adjacent exons are ligated together ultimately makes A site closer to splicing site Splicing/polyadenylation factors associate with phosphorylated CTD (Carbon terminal domain) of RNA polymerase; organizes factors for capping, intron removal • Ribozyme: An RNA molecule that functions as a catalyst in cellular reactions; Selfsplicing implies that RNA has catalytic activity, like an enzyme New riboenzymes can be created by synthesizing random chains of RNA, but their functionality is a question; used of an assay (= testing of catalytic activity)
Ability to bind to amino acid lysine and tRNA means that a riboenzyme identified have function equivalent of aminoacyl tRNA synthetases. Riboenzymes used in AIDs treatment by stopping the ADIS mRNA by binding AIDS mRNA & cleaving it or creation of AIDS proteins stopped by riboenzyme binding to AIDs protein & cleaving it. • Small interfering RNA (siRNA): 21 23 nucleotide, double stranded fragments formed when double stranded RNA initiates DNA silencing • RNA Silencing: A process where small noncoding RNA (derived from longer dsPrecursors) trigger sequence specific inhibition of gene expression Jorgensen flower pigment: Light purple flower expected to be more purple when added more purple pigments but observed is an albino flower, both endogenous and added genes were expressed but RNA encoded were destroyed = post transcriptional gene silencing. • Micro RNA (miRNA): Small RNA 2023 nu, synthesized from many sites in the genome and involved in inhibiting translation or increasing degradation of complementary mRNA Small interfering RNAs (siRNAs) and microRNAs (miRNAs) play roles in development and defense by mediating destruction of dsRNA or inhibiting translation Formation & mechanism of action siRNA miRNA; bobby pin 1 Origin: dsDNA is Origin: ssprecursor RNA cleaved by that contains complementary endonucleases dicer to sequences that allow them to small siRNA, over fold into pseudo dsRNA (pri hanging ends miRNA), cleaved by endonuclease Drosha at terminal c==* VAL Ł To elucidating the genetic code: 1) A new tool; In vitro translation system 1. Lyse cells = centrifuge to remove membranes 2. Addition of radioactive amino acids with RNA = radio active protein [ ] ^ 2) An organic chemist; Org. synthesis of polynucleotides 1. Bacterial extract with synthetic mRNA makes polypeptides, = 3/64 triplets 3) Brute force 1. Organic synthesis cannot control the ratios of nucleotides made, the relative [ ] of triplets is statistically determined, analyzed amino acid content of the protein produced by this mixture Random copolymer UUUG Eg. Codon: p(UUU) = 3*(¾) = 27/64 of getting UUU 4) Brilliance 1) Kgorana discovered how to synthesize defined triplets 2) Nirenberg & Ledger: filter binding assays I. Set up 20 cell free reactions each with 1 radioactive amino acid and 19 unlabelled amino acids II. Give each with same defined triplet III. Filter, to determine which is radioactive IV. Bound amino acid + trinucleotide retained in filter, in all other cases the AA* will go through All triplets decoded this way • Codon: Sequence of a nucleotides triplet in mRNA that specify amino acids the ‘genetic code’ is a triplet code, with each triplet set of nucleotides in RNA decoded into an amino acid. Three of the 64 codons are ‘stop’ codons that signal the end of the polypeptide chain. • Anticodon: A three nucleotide sequence in each tRNA that functions in recognition of the complementary mRNA codon • Transfer RNA (tRNA): small RNAs (~75AA) that translates the info encoded in nucleotide alphabet of an mRNA into amino acid alphabet of a polypeptide, tRNAs = adaptors. The anticodon of a tRNA forms complementary base pairs with the triplet codon in mRNA. tRNA brings proper amino acid to ribosome The anticodon site for amino acid attachement is at the 5’ end on the amino acid acceptor stem • Wobble hypothesis: Crick’s proposal that the steric req between the anticodon of the tRNA and the codon of
the mRNA is flexible at the 3rd position = only allow two codons that differ at 3rd to share the same tRNA during protein synthesis Often the 5’ nucleotide of tRNA anticodon will base pari with any 3’ nucleotide of the codon = same tRNA > 1codon Wobble base: Codon/Anti codon Anticodon Codon (wobblebase) U A or G G U or C I inosine derived from G U, C or A • Amino acyl tRNA synthetase (AARS): An enzyme that recognizes and covalently link amino acids to the 3’ ends of their equivalent tRNA, each amino acid is recognize by specific AARS, Catalyze two step reaction joining AA + tRNA Amino acid activation: ATP + AA AAAMP + PPi tRNA charging: AAAMP + tRNA AAtRNA + AMP Mechanism for translation: 1. Initiation codon: AUG, the site where ribosome attaches to the mRNA at assure that the ribosome is in the proper reading frame to reach entire msg or a missence protein is produced. In Eukaryotes Initiator tRNA finds mRNA 5’ cap structure recognized by initiation factor & binds, multicomplex formed and scans til identifies start codon, consensus sequence around AUG 2. Elongation: Cycles of elongation add amino acids to the growing chain, ribosome must move relative to mRNA Tu are elongation factors that bring AAtRNA to ribosome when bound to GTP = TGDP Ts are elongation factors that sponsors exchange of GTP for GDP on Tu = reactivates Tu TuGTP + AAtRNA AAtRNA to ribosome Process of elongation APE sites: a) At site A peptide bond formation by Peptidyl transferase: [Portion of the large ribosomal subunit that is responsible for catalyzing peptide bond formation] Peptide bond formation when (Psite)OH + H (Asite) H2O (water is released) to create single bond between amino & carboxy group b) Translocation: [move (a portion of a chromosome) to a new position on the same or another chromosome] occurs moving peptidyl tRNA to P site, GTP req c) A Site is empty, ready for next codon decoding d) Release of uncharged tRNA from E Site 3. Termination: Decoding of stop codon (3) as a termination signal, release of mature polypeptide No tRNA to decode = stop Release factors bind, GTP hydrolysis Complete polypeptides are released Ribosomes are dissociates from mRNA Overall for translation need: mRNA (template for codon), functional ribosome (decoding, moving), amino acyltRNA (Adapt and read codon, bring AA), energy (driving) In prokaryotes, a ShineDalgarno sequence 5’ to the AUG start codon in mRNA 1. Small ribosome subunit & initiation factors req, GTP required, tRNA 2. A,P,E sites: The ribosome has 3 functional sites
A = Aminoacyl/ Acceptor P = Peptidyl E = Exit
Incoming AAtRNA bind Growing polypeptide is held in place Uncharged tRNAs are released
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Polysome (polyribosome): The complex formed by an mRNA & a number of ribosomes in the process of translating that mRNA Implicates that many copies of a protein can be produced from a single mRNA
Lecture 9: Nuclear & chromatin structure
