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SURVEY OF BIOCHEMISTRY Transcription and Translation 1 PRS Which of the following is not an example of a DNA-binding motif in proteins? 1.Beta Sheet 2.Helix-turn-Helix 3.Zinc Finger 4.Leu Zipper 2 PRS • Which amino acid contains a hydroxyl in its side chain? 1. Threonine 2. Methionine 3. Aspartic Acid 4. Phenylalanine 3 Transcription Transcription is the process by which RNA is produced from a DNA template. Steps involved in transcription Initiation Elongation Termination 4 Transcription DNA has to separate at the position where transcription occurs. Fig. 26-3 5 Transcription Sense Strand Newly synthesized RNA has the same general sequence as the sense strand; U in RNA, T in DNA 5’-…GATCCGTAGGTC…-3’ 3’-…CTAGGCATCCAG…-5’ Antisense Strand (template) 5’-…GATCCGTAGGTC…-3’ 5’----GAUCCGUAGGUC…3’ 3’-…CTAGGCATCCAG…-5’ 6 Transcription Initiates at Promoter Sequences What is a gene? Example of E.coli Lactose Operon Organization Fig. 26-4 P: Promoter O: Operator I: Inhibitor 7 Structural genes follow promoter sequences In E. coli, the lactose operon consists of 3 structural genes: Beta-Galactosidase (Z) Galactoside Permease (Y) Thiogalactoside Transacetylase (A) 8 E. coli Promoter Sequences Notice how sequence is numbered! Usually single purine 9 Prokaryotic RNA Polymerases 2 ' Core enzyme The sigma factor binds specifically to the promoter. Core enzyme affinity to DNA ~5x10-12 M. Holoenzyme = sigma factor 10 Elongation: RNA Chain Growth 3’-OH group acts as a nucleophile, attacking the alpha phosphate group, with subsequent release of inorganic pyrophosphate. 11 DNA Supercoiling Negative supercoiling Positive supercoiling 1. RNA polymerase is processive 2. Once an RNAP has moved from the promoter, another can bind 3. Transcription is fast: constitutive enzymes vs. inducible enzymes 12 Termination of Transcription Because the newly synthesized RNA is single-stranded, hairpin structures can easily form from the termination sequence. 13 Hairpin Structure in RNA G-C rich bp region What happens when a known terminator sequence is missing? Poly-U tail 14 Termination: Rho Factor Helicase: RNA-RNA and RNA-DNA complex unwinding. 15 Transcription in Eukaryotes • RNA Polymerase I – Precursors to rRNA • RNA Polymerase II – Precursors to mRNA • RNA Polymerase III – Precursors to 5S rRNA, tRNA – Precursors to other small RNA’s 16 Eukaryotic Transcription Factors Transcription factors are req’d to initiate transcription, and facilitate elongation in eukaryotes. Eukaryotic Termination: Not well understood 17 Portion of the Genetic Code Wobble 18 Reading Frames 19 Translation: Protein Synthesis • Messenger RNA – Template • Transfer RNA – Carries amino acids • Ribosomal RNA – Aids protein synthesis 20 tRNA Structures 2D 3D 21 tRNA’s have modified bases Be able to name, draw, recognize structures from Figure 27-4. 22 Aminoacyl tRNA Synthetases Aminoacyl tRNA synthetases add aa’s to tRNA. Step 1. 23 Aminoacyl tRNA Synthetases Step 2. + tRNA 24 Ribosome 25 Sites in the Ribosome A Aminoacyl E P A P Peptidyl E Exit 26 Chain Initiation in Translation Start Codon: AUG Methionine 27 Shine-Dalgarno Sequence upstream downstream 28 Initiation 29 30 Translation: Polypeptide Synthesis Peptidyl Transferase Activity 31 Elongation 32 Administrative Updates • Exam 1 on Thursday, 4-5 pm – 20 questions – Short answers and Multiple Choice – Closed-book – Calculators allowed – No cell phones, blackberries, etc. • Office Hours: M, T, R 2-4 pm 33