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

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slide1

Announcements

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

Specifics on reading assignments: Ch. 11: Skip, p. 304, btm. 309- top 312; Ch. 12: skim 327-328; skip btm 335-336; skip recombination on 338-341; Ch. 13:

slide2

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

slide3

Outline of Lecture 23

The Genetic Code - biochemical evidence

Transcription

slide4

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-...
slide5

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-...
slide6

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.
slide7

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.
slide9

Degeneracy and the Wobble Hypothesis

  • Codon in mRNA
  • Anticodon in tRNA
  • Codon: 5’-1-2-3-3’
  • Anticodon: 3’-3-2-1-5’
  • 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”)
slide11

II. TranscriptionMaking Sense of the Strands

  • DNA coding strand = Sense Strand
  • DNA template strand = Antisense Strand
  • mRNA formed = Sense Strand

Coding strand

5’

3’

mRNA 5’

3’

5’

3’

Template strand

slide12

Prokaryotic Promoter Lies Just Upstream (5’) of Transcribed Region; RNA Polymerase Binds Two Places

-35 Region

-10 TATA Box

slide13

Initiation of Prokaryotic Transcription Requires Binding of Sigma Factor to Pol

Note: No primer needed

5’ to 3’

slide14

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.
slide15

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

slide16

Results of Experiment

[NaCl]

Total

Protein

RNA

Synthesis

Activity

RNA

Synthesis

+ 1 ug/ml

 amanitin

I

II

III

0 10 20 30 40 50

Fraction #

slide17

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.
slide18

Anatomy of a Eukaryotic Gene (Protein Encoding)

Pol II, Basal TFs bind

CAAT Box

TATA Box

Cis-regulatory Elements may be located thousands of bases away; Regulatory TFs bind.

slide19

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
slide20

TF

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

slide21

RNA Processing in Eukaryotes

Pre-mRNA (primary transcript)

5’ cap

Poly A tail

Splicing

Mature mRNA