Chapter 30 (Lecture 17-21) – Review Quicksheet

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Margaret Fahnestock

Biochem 2B03 2012 Quicknotes  mRNA sequence composed of triplets  Shine-Delgarno Site o Purine-rich sequence near 5’ end of transcript o 4-8 nt upstream of start codon (AUG; Met) o Recruits ribosome Acceptor Stem – attaches to tRNA amino acid CCA 3’  Unusual bases found in tRNA and rRNA o Pseudouridine (ψ), ribothymidine (T), Dihydrouridine (D) D-loop – contains  Secondary structural elements – stems, loops, bulges, and junctions dihydrouridine; variable (regions where several stemloop structures meet) length 7-12 nt Tψc loop – contains  Structural motifs – U-turns, tetraloops and bulges pseudouracil (ψ)  Tertiary structural motifs – arise from coaxial stacking, pseudoknot contains thymine formation and ribose zippers  Aminoacyl tRNA – bridges the genes and proteins; recognizes codons, positions amino acids in ribosome for peptide bond formation o Amino acid added to 3’ end of tRNA (at CCA) Variable Loop –  Aminoacyl tRNA Synthetase (AAS) – mediates amino acid attachment present in some to tRNA  Amino acylation of tRNA (two steps) tRNAs Anticodon – recognizes codon 1. Formation of the aminoacyl-adenylate – “activated” form of amino acid 2. Transfer of the activated amino acid to the 2’-OH or 3’-OH of the tRNA (Class I and Class II AAS mechanisms) o Class I AAS – adds amino acid to 2’-OH of tRNA; later transesterification changes it to 3’OH o Class II AAS – adds amino acid to 3’-OH of tRNA o Class Rule – each amino acid is loaded onto tRNA by either Class I or Class II enzyme  Exception – lysine in some Archaea  tRNAs matched to cognate amino acids by AAS – interprets 2dgenetic code (structural feature/recognition elements); not universal o Anticodon loop (at least 1 or 3 bases) o Acceptor stem (CCA 3’) o Position 73 (unpaired base preceding the CCA/acceptor end) o Variable loop o EXCEPTION  tRNA Ala– non-W/C base pair (G:U) at position 70; essential determinant of the tRNA/AAS match  AAS prevent mis-charging of tRNA when amino acid structure is similar – tRNA charging accuracy Tyr o Tyr and Phe – tRNA AAS does not charge Phe because stable binding by the amino acid activating site (reaction 1) requires OH group in Tyr 1 Biochem 2B03 2012 o Val and Ile – at [Val]=5x[Ile] ratio, tRNA AAS binds Val 3% of the time; Val-tRNA enters proof-reading site in tRNA AAS Ile Ile where hydrolysis removes incorrect amino acid from tRNA (correctly charged Ile-tRNA does not fit in this site due to extra methyl group on side chain) o Val and Thr – acylation site of tRNA Valis hydrophobic and favours valine (hydrophobic); proof reading site of tRNA ValAAS is hydrophilic (favours interaction with threonine) Ribosomes (E. Coli) – 70S  Roughly 2/3 RNA  20,000 ribosomes in a cell; 20% of cells mass  rRNA encoded by 7 rrn operons and processed post-transcriptionally by RNase II  Small Subunit o 30S; made up of 21 proteins and a 16S rRNA o mRNA binding, “decoding center”, codon/anticodon base pairing  Large subunit o 50S; made up of 31 proteins and a 23S rRNA and a 55S rRNA o Catalysis of peptide bond formation; “peptidyl transferase center” Catalytic Center of Ribosome – rRNA, not protein  Found by Noller Lab; 1 treatment of ribosomes with proteases (some activity retained), 2 treatment of ribosomes with
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