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Chapter 12

Bio 1090 ch.12.docx

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University of Guelph
BIOL 1090
Andrew Bendall

Bio 1090 Chapter 12 p.293-294, p. 298-305, p. 306-312 Translation and the genetic code Protein synthesis: translation - Genetic information in mRNA is translated into amino acid sequences of polypeptide according to the specifications of genetic code - Require large number of macromolecules 1. + 50 polypeptides and 3-5 RNA molecules present in each ribosomes 2. 20 amino acid-activating enzymes 3. 40-60 tRNA molecules 4. + soluble proteins involved in polypeptide chain initiation, elongation and termination (present in large quantities translation system makes up a major portion of the metabolic machinery) Overview of protein synthesis - Translation process : 1. Gene expression; transcription involves the transfer of info stored in genes to messenger RNA and carry info to sites of polypeptide synthesis in cytoplasm 2. Translation; involves the transfer of info in mRNA molecules into the sequences of amino acids in polypeptide gene products. Occurs on ribosomes; complex macromolecular structures in cytoplasm. - Involve 3 type of RNA; transcribed form DNA templates - 3-5 RNA molecules present in ribosome and 40-60 small RNA function as adaptors by mediating incorporation of proper amino acids into polypeptides in response to specific nucleotide sequences on mRNAs - Amino acids attached to tRNA by set of activating enzymes; aminoacyl-tRNA synthetases - Nucleotide sequence of mRNA translated into amino acid sequence according to gene code (some contain short amino acid sequences at amino or carboxyl termini that function as signal for transport into cellular some compartment) - nascent secretory proteins; short signal sequence at amino terminus that directs emerging polypeptide to ER - some nuclear protein contains targeting extensions at carboxyl termini (some are removed by peptidases) - ribosomes are non specific; can synthesize any polypeptide encoded by mRNA (each mRNA translated by several ribosomes; formation of polyribosome or polysome) Translation: the synthesis of polypeptide using mRNA templates - mRNA provide specification for amino acid sequences of polypeptide gene products - ribosomes provide macromolecule components for translation - tRNAs provide adaptor to incorporate amino acids into polypeptides in response to condons in mRNAs Translation: polypeptide chain initiation - include events proceeding formation of peptide bond between 2 amino acids of new polypeptide chain - In E. coli, initiation involves 30 S subunit of ribosome; initiator tRNA, mRNA, 3 soluble protein initiation factors IF- 1, IF-2 and IF-3, and 1 molecule of GTP - Occurs at 70S ribosome, but it dissociates into 30 S and 50 S subunits when they complete synthesis of polypeptide chain - 1 stage: free 30 S subunits interact with an mRNA and initiation factors. 50S subunit joins complex to form 70 S ribosome in final step of initiation - Synthesis of polypeptide initiated by tRNA (tRNA Met) in response to initiation codon l (AUG or GUG), begin with methionine, amino-terminal methionine cleaved from polypeptides functional protein don’t have amino-terminal methionine - Methionine on tRNA l Methas amino group blocked with formyl group - Distinct methionine responds to internal methionine codons; both methionine tRNAs have same anticodon and respond to same codon (AUG) - Only methionyl-tRNA fMet interacts with protein initiation factor IF-2 to begin initiation - Only tRN fMetbinds to ribosome in response to AUG initiation codons in mRNAs leaving Met methionMettRNA bind in response of internal AUG codons - tRNA f binds to ribosomes in response to alternate initiator codon GUG that occurs in mRNA molecules - polypeptide chain initiation begins with formation of 2 complexMet 1. contains initiation factor IF-2 and methionyl-tRNA f 2. contains mRNA molecule, 30S ribosomal subunit and initiation factor IF-3; controls ability of 30 S subunit to begin initiation - formation of 30S depends on base-pairing between nucleotide sequence near 3’ end of the 16 S rRNA and sequence near 5’ end of mRNA - prokaryotic mRNAs contain polypurine tract located 7 nucleotides upstream from AUG initiation codon; shine-dalgarno sequence (near 3’ terminus and 16S ribosomal RNA), when its modified, the modified mRNAs either are not translated  base-paring plays role in translation - IF-2 and IF-3 complex combine with each other and with IF-1 and GTP to form 30S initiation complex - Final step: addition of 50S subunit to 30S initiation complex to produce 70S ribosome - IF-3 must be released and 50S subunit never associated with 30 S subunit at same time (addition of 50S requires energy from GTP and release ofMet-1 and IF-2) - Addition of 50 S position tRNA, methionyl-tRNA f in P site with anticodon of tRNA with AUG initiation codon of mRNA - Methionyl-tRNA Metonly aminoacyl-tRNA that can enter P site without passing through f nd aminoacyl (A) site. ndth AUG at P site, 2 codon of mRNA register with the A site; setting stage for 2 phase in polymerase chain elongation - Similar in eukaryote except: (1) amino group of methionine on initiator tRNA is not formylated (2) initiation complex forms at the 5’ terminus of mRNA not at shine-dalgarno/AUG translation start site - initiation complex scans the mRNA starting at 5’ end searching for AUG translation initiation codon (initiation starts at AUG closest to 5’ terminus; efficiency depends on contiguous nucleotide sequence) - optimal initiation sequence is 5’–GCC(A or G)CCAUGG-3’ - purine (A or G) 3 bases upstream from AUG initiator codon and G important  influencing initiation efficiency by tenfold + - changes in other bases causes smaller decrease in initiation efficiency ** Kozak’s rules Met - eukaryoMetcontain special initiator tRNA; tRNA i but amino group of methionyl- tRNA i is not formylated (interacts with soluble initiation factor and enter the P site during initiation) - in eukaryote, cap-binding protein binds to 7-methyl guanosine cap at 5’ terminus of the mRNA - initiation complex moves 5’3’, searching for AUG codon - AUG triplet found- initiation factors dissociate from complex and large 60 S subunit binds to methionyl-tRNA/mRNA/40S, forming 80S ribosome  elongation Translation: polypeptide chain elongation - addition of each amino acid to growing polypeptide occurs in 3 steps (1) binding of an aminoacyl-tRNA to the A site of the ribosome -3 nucleotides in the anticodon of incoming aminoacyl-tRNA must pair with nucleotide of mRNA codon at A site -requires elongation factor Tu carrying a molecule of GTP (GTP required for aminoacyl- tRNA binding at A site but not cleaved till peptide bond formed (after cleavage, EF-Tu- GDP released from ribosome- inactive but converted to active EF-Tu-GTP by elongation factor Ts which hydrolyze 1 molecule of GTP) -EF-Tu interacts with all aminoacyl-tRNAs except methionyl-tRNA (2) transfer of the growing polypeptide chain form the tRNA in the P site to the tRNA in the A site by formation of a new peptide bond -uncouples the growing chain from the tRNA in the P site and covalently joins chain to the tRNA in A site  catalyzed by peptidyl transferase; enzymatic activity built into 50S of ribosome -peptidyl transferase activity resides in 23S rRNA rather than in ribosomal protein -peptide bond formation requires hydrolysis of the molecule of GTP brought by ribosome in EF-Tu (3) translocation of the ribosome along the mRNA to position the next codon in the A site. Nascent polypeptide-tRNA and uncharged tRNA are translocated from A and P sites to the P and E sites as ribosome moves 3 nucleotides toward 3’ end of mRNA -requires GTP and elongation factor G (EF-G), ribosome undergoes changes in conformation during translocation; may shuttle along the mRNA energy for movement of ribosome provided by hydrolysis of GTP -translocation of peptidyl-tRNA from A site to P site leaves A site and ribosome - protein fold up on the surface of the ribosome during synthesis, but fibroin remains extended from surface of ribosome  nascent polypeptide chains of increasing length can be seen attached to ribosomes as they scanned for 5’ end of mRNA to 3’ end - fibroin is large protein; synthesized on large polyribosomes containing 50-80 ribosomes - in E. coli; 3 steps required to add one amino acid to the growing polypeptide chain occurs in about 0.05 sec Translation: Polypeptide chain termination - when any of 3 chain-termination codons (UAA, UAG or UGA) enters A site on ribosome; recognize by soluble proteins; release factors - in E. coli, 2 release factors, RF-1 (recognizes termination
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