Announcements 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: skim btm 353-354; 358-top 360; skip splicing mechanism, editing, and EM (367-370). Problems to look over: Ch. 11: 4, 5, 8, 15; Ch. 12: 3, 10, 21, 22; Ch. 13: 2, 6, 7, 13, 15, 23. Homework due next Friday, 11/1. Last day to withdraw is next Fri. Nov. 1. If you have concerns, talk to me.
Review of Last Lecture Small group discussion on Watson-Crick paper The Genetic Code - biochemical evidence for triplet code
Outline of Lecture 24 I. tRNA and the genetic code II. Transcription - prokaryotes III. Transcription - eukaryotes IV. RNA Processing
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 The genetic code is nearly universal. Exceptions: yeast mitochondria Tetrahymena Mycoplasma
Degeneracy and the Wobble Hypothesis C • 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”) G U G
II. Transcription: RNA from DNA What is the enzyme that can direct RNA synthesis? RNA polymerase - first isolated in liver of rats - requires NTPs with ribose as sugar - NO primer is needed DNA n(NTP) (NMP)n + n (Ppi) enzyme RNA polymerase well characterized in E. coli - 4 subunits
Making Sense of the Strands • DNA coding strand = Sense Strand • DNA template strand = Antisense Strand • mRNA formed = Sense Strand Coding or Partner strand 5’ 3’ mRNA 5’ 3’ 5’ 3’ Template strand
Components of Prokaryotic Transcription 3 main steps of transcription: initiation, elongation, termination
NO primer required 5’ to 3’ 50 nt/second
Prokaryotic Promoter Lies Just Upstream (5’) of Transcribed Region Two Consensus sequences -35 Region -10 TATA Box Effect of mutations here?
Termination of Transcription in Prokaryotes • A specific nucleotide sequence acts as a termination signal, about 40 base pairs in length • Sometimes a special protein called termination factor, rho is required for termination • At termination, RNA dissociates from DNA and enzyme (RNA polymerase) falls off too
III. Isolating Eukaryotic RNA PolymerasesTranscription in euk. much more complex • Roeder and Futter (1974): Are there enzymes in the eukaryotic 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
Results of Experiment [NaCl] Total Protein RNA Synthesis Activity I II III 0 10 20 30 40 50 Fraction # How many euk. RNA polymerases are there?
Eukaryotic RNA Polymerases • RNA Pol I • makes three rRNAs (28S, 18S, 5.8S) • RNA Pol II - makes mRNA and snRNA (small nuclear RNA) • RNA Pol III • makes tRNA and 5S rRNA • How does each pol know which DNA to bind? Each recognizes different core promoter regions.
Anatomy of a Eukaryotic Gene Pol II, Basal TFs bind CAAT Box TATA Box Cis-regulatory Elements may be located thousands of bases away; Regulatory TFs bind.
Binding of Eukaryotic RNA Pol II Requires Binding of Basal TFs to Core Promoter TF
RNA Processing in Eukaryotes STABILITY
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, no processing in prokaryotes