Beadle and Tatum Experiment
Lecture 4 Intrdocution to Transcription Rrna Synthesis and Maturation SP. Figs 11-4,6,7,9,12,13,14 - Beadle and Tatum Experiment (1940s) Support for “1 gene/1 enzyme” hypothesis B&T receive Nobel Prize in 1958 Second Picture: Demonstrates deficiency in vitamin biosynthesis 3RD Picture: Mutation in gene encoding protein involvedin making vitamin pantothenic acid
Generic Biochemical Pathway
If Enzyme #2 gene mutated, the providing cells w/ C will ‘rescue’, but roviding A and B will not. B&T proposed, “1 gene-1 enzyme” hypothesis Eukaryotic Transcription Steps: 1. 51 TO 31 (strand being synthesized) 2. Unwinnding DNa = topological stress, supercoiling alleviates torsional stress 3. NTP ydrolysis provides energy 4. Many polymerases can act simultaneously Prokaryotic Trsnacription Random initiation sites initiation occurs at proper sites Sigma factor binds to seqs. At -10/-35 Arrangments of elements in bacterial promotor – consensus sequences ocated @-35 and -10 in prokaryotic genes
3 different eukaryotic RNA polymerases can be distinguished by their susceptibility to alpha-amanitin Ribosomes: Several rRNAs and many proteins Many copies of the Rrna GENES Nucleolus: ‘Clusters; of Rrna genes And site of ribosome assembly Transcription of Rrna genes by multiple polymerases Many eukaryotes: Rrna gene clusters (NOR) exist as tandem repeats on several x-somes RNA size can be estimated based on sedimentation of molecule during centrigation S/Svedberg value: Correlated with size and shape, so larger S value means that the molecule is larger (with perhaps a greater SA) than one have a lower S value Primary Rrna transcripts are 45S, processed to 28S 18S, 5.8S Note: S values are generally not additive; that is no equal to 45s molecule form which they arise The Pulse Chase Experiment 1. Pulse: Add radioactive precursor to cells for short peiod. Get incorporation of label in macromolecule pool 2. Chase: Wash cells to remove radioactive precursor, then examine radioactive macromolecules after on incubation period Utility: A basic biochemical tool to “tag” a molecule of population of molecules, and determine their fate over a time course Examples: 1. Did the molecules become smaller (processing degradation) 2. Did molecules move? (i..e from cytoplasm to nucleus) Penman Experiment How is Rrna made and processed and where do these events occur? 1. Label cells for short period/pulse 2. Was haway label. Start incubation/chase 3. Prepare nucleolar and cytoplasmic fractions 4. Isolate RNA from these fuctions 5. Spearate the RNA by size (centrifugation) 6. Determine the distribution of RNA molecules and follow the fate of the initial radiolabelled molecules Top: Nucleolar fractions Bottom: Cytoplasmic fractions Blue Lines: All RNA (absorbace measurement) Red Lines: Pulse labelled RNA (radioactivity measurement) Concllude: After 10 chase most RNA is 45S, and still nucleolar fraction
Generic Biochemical Pathway
If Enzyme #2 gene mutated, the providing cells w/ C will ‘rescue’, but roviding A and B will not. B&T proposed, “1 gene-1 enzyme” hypothesis Eukaryotic Transcription Steps: 1. 51 TO 31 (strand being synthesized) 2. Unwinnding DNa = topological stress, supercoiling alleviates torsional stress 3. NTP ydrolysis provides energy 4. Many polymerases can act simultaneously Prokaryotic Trsnacription Random initiation sites initiation occurs at proper sites Sigma factor binds to seqs. At -10/-35 Arrangments of elements in bacterial promotor – consensus sequences ocated @-35 and -10 in prokaryotic genes
3 different eukaryotic RNA polymerases can be distinguished by their susceptibility to alpha-amanitin Ribosomes: Several rRNAs and many proteins Many copies of the Rrna GENES Nucleolus: ‘Clusters; of Rrna genes And site of ribosome assembly Transcription of Rrna genes by multiple polymerases Many eukaryotes: Rrna gene clusters (NOR) exist as tandem repeats on several x-somes RNA size can be estimated based on sedimentation of molecule during centrigation S/Svedberg value: Correlated with size and shape, so larger S value means that the molecule is larger (with perhaps a greater SA) than one have a lower S value Primary Rrna transcripts are 45S, processed to 28S 18S, 5.8S Note: S values are generally not additive; that is no equal to 45s molecule form which they arise The Pulse Chase Experiment 1. Pulse: Add radioactive precursor to cells for short peiod. Get incorporation of label in macromolecule pool 2. Chase: Wash cells to remove radioactive precursor, then examine radioactive macromolecules after on incubation period Utility: A basic biochemical tool to “tag” a molecule of population of molecules, and determine their fate over a time course Examples: 1. Did the molecules become smaller (processing degradation) 2. Did molecules move? (i..e from cytoplasm to nucleus) Penman Experiment How is Rrna made and processed and where do these events occur? 1. Label cells for short period/pulse 2. Was haway label. Start incubation/chase 3. Prepare nucleolar and cytoplasmic fractions 4. Isolate RNA from these fuctions 5. Spearate the RNA by size (centrifugation) 6. Determine the distribution of RNA molecules and follow the fate of the initial radiolabelled molecules Top: Nucleolar fractions Bottom: Cytoplasmic fractions Blue Lines: All RNA (absorbace measurement) Red Lines: Pulse labelled RNA (radioactivity measurement) Concllude: After 10 chase most RNA is 45S, and still nucleolar fraction
