Announcements 1. Specifics on reading assignments: Ch. 11: Skip, p. 304, btm top 312; Ch. 12: skim ; skip btm ; skip recombination on ; Ch. 13: skim btm ; 358-top 360; skip splicing mechanism, editing, and EM ( ). 2. Problems to look over: Ch. 11: 4, 5, 8, 15; Ch. 12: 3, 10, 21, 22; Ch. 13: 2, 6, 7, 13, 15, Homework due next Friday, 11/1. 4. Last day to withdraw is next Fri. Nov. 1. If you have concerns, talk to me.

Review of Last Lecture 1. Small group discussion on Watson-Crick paper 2. 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 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”) G C G U

Special Anticodon-Codon Base-Pairing Rules

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 n(NTP) (NMP) n + n (Pp i ) DNA 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 3’ 5’3’ 5’ mRNA 5’3’ Coding or Partner strand 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 -10 TATA Box-35 Region Two Consensus sequences 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 Polymerases Transcription 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 Fraction # [NaCl] Total Protein RNA Synthesis Activity I II III 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 TATA BoxCAAT Box Cis-regulatory Elements may be located thousands of bases away; Regulatory TFs bind. Pol II, Basal TFs bind

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

RNA Processing in Eukaryotes STABILITY

Introns and Exons

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