Post on 14-Dec-2015
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
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.
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”)
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