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. 309- top 312; Ch. 12: skim 327-328; skip btm 335-336; skip recombination on 338-341; Ch. 13:

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

Outline of Lecture 23

I. The Genetic Code - biochemical evidence

II. Transcription

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-...

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-...

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.

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.

Note that 3rd Base Position is Variable

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”)

Special Anticodon-Codon Base-Pairing Rules

II. TranscriptionMaking 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

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

-10 TATA Box-35 Region

Initiation of Prokaryotic Transcription Requires Binding of Sigma Factor to Pol

5’ to 3’

Note: No primer needed

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.

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 negativecharge; some proteins bind strongly, most don’t.

Add nuclei, containingproteins

Elute with Na+ gradient

Results of Experiment

Fraction #

[NaCl]

TotalProtein

RNASynthesisActivity

RNASynthesis+ 1 ug/ml amanitin

0 10 20 30 40 50

I

II

III

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.

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

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

Binding of Eukaryotic RNA Pol II Requires Binding of Basal TFs

to Core Promoter

TF

RNA Processing in Eukaryotes

Pre-mRNA (primary transcript)

5’ cap

Poly A tail

Splicing

Mature mRNA

Introns and Exons