ICE 4 Review Translation and Gene regulation Marty and Aaron October 25th, 2014

Translation

From genes to proteins

Two enzymes involved in translation amino acyl-tRNA synthetases ribosome Site where tRNAs act as adaptors between mRNA codons and amino acids Proofreads codon-anticodon pairing Catalyzes peptidyl-transferase reaction Adds amino acids to the 3’ of a tRNA Proofreads that appropriate tRNA is attached

aa-tRNA synthetases Both steps catalyzed at the synthesis site: Adds amino acids to 3’ adenine of tRNA Cleaves amino acids off of 3’ adenine of tRNA

Ribosomal Elongation A- site: where amino-acylated tRNAs are deposited by EF-Tu when appropriate codon-anticodon pairing occurs P-site: where the tRNA is bound to a polypeptide as opposed to a single amino acid E-site: where a spent tRNA goes before getting kicked out

Peptide bond formation The peptide bond is formed between the free amino group of the amino acid in the A site and the carboxyl group at the end of the polypeptide chain in the P site. The reaction is driven by the high energy of the acyl linkage between the carboxyl group and the 3’ hydroxyl at the end of the tRNA and is catalyzed by the large subunit of the ribosome. Thus, the energy for peptide bond formation is derived from the expenditure of a molecule of ATP in the charging of tRNA. Lone pair of electrons from the amino group nitrogen in A attacks carboxyl carbon in P.

Elongation Factors (“EF”s) We learned about two elongation factors used by the ribosome, both are enzymes that hydrolyze GTP (i.e., “GTPases”) EF-Tu uses GTP/GDP to switch between two conformations: When bound to GTP, EF-Tu tightly binds a aa-tRNA When bound to GDP, EF-Tu binds aa-tRNA much more weakly This is why EF-Tu has to hydrolyze GTP to release an aa-tRNA into the ribosome’s A-site EF-G uses the energy released by hydrolyzing GTP to push the tRNAs and the mRNA in the ribosome over by one codon

Elongation Factor (EF)-Tu deposits aa-tRNAs into the A-site

Elongation Factor (EF)-G translocates the ribosome after the peptidyl- transferase reaction

Transcription/translation question a) How many amino acids does this DNA sequence code for? 7 b) How many amino acids does this sequence code for if C18 is mutated to G? 4 c) How many amino acids does this sequence code for if G20 is deleted? 5

Transcription/translation question 3’ a) Please write the first 18 bases of the mRNA that would be transcribed, 5’ to 3’. 5’-AGGUCCACGGGUCCACCG-3’ b) What are the first five amino acids of the protein derived from the mRNA (please denote the N- and C-terminal ends of the peptide) N-Met- Thr- His- Ile- Val- C (ATG) (ACT) (CAC) (ATC) (GTT) c) What are the last five amino acids of the protein derived from the mRNA (please denote the N- and C-terminal ends of the peptide) N-Ser- Gly- Ile- Gln- His- C (AGC) (GGC) (ATC) (CAG) (CAT) (TAA = stop)

Mechanistic Ribosome Question from a pset EF-Tu will hydrolyze a GTP and deposit an aa-tRNA in the A site. codon: 5’-GUG-3’  valine codon: 5’-GAU-3’  aspartic acid

Given the progress of the ribosome along the mRNA diagrammed below, label the 5’ and 3’ ends of the mRNA along with the A-, P-, and E-sites 5’ E P A E 3’ Label whether the N- or C-terminus of the growing polypeptide chain is emerging from the ribosome N-terminus