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Announcements 1. Grading of Mendel papers: A= completeness of answer (40); F = flow and organization (15); R = references (10); S = spelling and grammar.

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Presentation on theme: "Announcements 1. Grading of Mendel papers: A= completeness of answer (40); F = flow and organization (15); R = references (10); S = spelling and grammar."— Presentation transcript:

1 Announcements 1. Grading of Mendel papers: A= completeness of answer (40); F = flow and organization (15); R = references (10); S = spelling and grammar (10). 2. Specifics on reading assignments: Ch. 11: Skip, p. 304, btm top 312; Ch. 12: skim ; skip btm ; skip recombination on ; Ch. 13:

2 Review of Last Lecture 1. Eukaryotic DNA replication is complex 2. The “end” problem and telomerase: aging and cancer 3. The Genetic Code - theoretical evidence for triplet code; genetic evidence using mutagens, ie. insertions and deletions can cause frameshift mutations

3 Outline of Lecture 23 I. The Genetic Code - biochemical evidence II. Transcription

4 I. Biochemical Evidence 1961: Nirenberg, Matthaei used synthetic mRNAs and an in vitro translation system to decipher the code. Polynucleotide Phosphorylase enzyme links NTPs to make RNA without a template Homopolymers: –poly(U) codes for Phe-Phe-Phe-Phe-… –poly(A) codes for Lys-Lys-Lys-Lys-… –poly(C) codes for Pro-Pro-Pro-Pro-...

5 Repeating Copolymers Khorana, early 1960’s UGUGUGUGUGUGUGUGU... – Cys-Val-Cys-Val-Cys-Val-... –Therefore GUG or UGU codes for either Cys or Val UUCUUCUUCUUCUUC… –Phe-Phe-Phe-Phe-... or –Ser-Ser-Ser-Ser-… or –Leu-Leu-Leu-Leu-...

6 In Vitro Triplet Binding Assay Nirenberg and Leder (1964) mixed all 20 amino acids with ribosomes, different RNA triplets: –Ribosomes + UAU -> Tyr binds –Ribosomes + AUA -> Ile binds –Ribosomes + UUU -> Phe binds, etc.

7 Nucleic Acid to Protein How does the information in codons of mRNA get translated into amino acids in polypeptides? Through adapter molecules: tRNA tRNA has anticodon that base pairs with the codon in mRNA and carries an amino acid corresponding to that codon.

8 Note that 3rd Base Position is Variable

9 Degeneracy and the Wobble Hypothesis Codon in mRNA Anticodon in tRNA Codon: 5’ ’ Anticodon: 3’ ’ First two bases of codon are more critical than 3rd base Base-pairing rules are relaxed between 3rd base of codon and 1st base of anticodon (third base “wobble”)

10 Special Anticodon-Codon Base-Pairing Rules

11 II. Transcription Making Sense of the Strands DNA coding strand = Sense Strand DNA template strand = Antisense Strand mRNA formed = Sense Strand 3’ 5’3’ 5’ mRNA 5’3’ Coding strand Template strand

12 Prokaryotic Promoter Lies Just Upstream (5’) of Transcribed Region; RNA Polymerase Binds Two Places -10 TATA Box -35 Region

13 Initiation of Prokaryotic Transcription Requires Binding of Sigma Factor to Pol 5’ to 3’ Note: No primer needed

14 Termination of Transcription in Prokaryotes Occurs when hairpin loops form from intramolecular GC base pairing in mRNA. Sometimes a special protein called termination factor, rho is required for termination.

15 Isolating Eukaryotic RNA Polymerases Roeder and Futter (1974): Are there enzymes in the nucleus that make RNA? From cultured frog cells, isolated nuclei. Separated proteins by Ion-Exchange Chromatography: Beads with negative charge; some proteins bind strongly, most don’t. Add nuclei, containing proteins Elute with Na + gradient

16 Results of Experiment Fraction # [NaCl] Total Protein RNA Synthesis Activity RNA Synthesis + 1 ug/ml  amanitin I II III

17 Eukaryotic RNA Polymerases RNA Pol I –1000  g/ml  -amanitin inhibits –makes three rRNAs (28S, 18S, 5.8S) RNA Pol II –1  g/ml  -amanitin inhibits –makes mRNA and snRNA (small nuclear RNA) RNA Pol III –50  g/ml  -amanitin inhibits –makes tRNA and 5S rRNA Each recognizes different core promoter regions.

18 Anatomy of a Eukaryotic Gene (Protein Encoding) TATA BoxCAAT Box Cis-regulatory Elements may be located thousands of bases away; Regulatory TFs bind. Pol II, Basal TFs bind

19 Eukaryotic vs. Prokaryotic Transcription In eukaryotes, transcription and translation occur in separate compartments. In bacteria, mRNA is polycistronic; in eukaryotes, mRNA is usually monocistronic. –Polycistronic: one mRNA codes for more than one polypeptide –moncistronic: one mRNA codes for only one polypeptide 3 RNA polymerases in euk., 1 in prok. Binding of Basal Transcription Factors required for euk. RNA Pol II binding. Processing of mRNA in eukaryotes: –5’ 7-methylguanosine (7mG) cap added –3’ Poly-A tail added –Splicing out of introns

20 Binding of Eukaryotic RNA Pol II Requires Binding of Basal TFs to Core Promoter TF

21 RNA Processing in Eukaryotes Pre-mRNA (primary transcript) 5’ cap Poly A tail Splicing Mature mRNA

22 Introns and Exons

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