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UVM BIOC 302 - Protein synthesis I

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Protein synthesis IBiochemistry 302Key features of protein synthesisPrimary steps involved in synthesizing a functional proteinSubunit composition of the prokaryotic ribosome (~2/3 rRNA, 1/3 protein)Ribosomal components: prokaryotes versus eukaryotesAssembly of small ribosomal subunit is an ordered process in vitroMonovalent & divalent cations modulate 70S ribosome assembly in vitroRibosomal subunits have distinct function roles in protein synthesisPutative secondary structure of E. coli 16S rRNAEarly EM studies reveal 3D topography of large and small ribosomal subunits3D-structure of small ribosomal subunit of Thermus thermophilus(shape determined by RNA component)3D-structure of small ribosomal subunit of Thermus thermophilus (decoding center made entirely of RNA)Not to be outdone…Steitz group determines crystal structure of 50S ribosomal subunit of Haloarcula marismortuiA representative prokaryotic mRNA: the lac operon (a polycistronic message)Initiation requires ribosomal alignment:Shine-Dalgarno sequencesEssential prokaryotic protein factors involved in translationInitiation of protein synthesis in prokaryotesA word about the prokaryotic initiator tRNA, fMet-tRNAfMetChemical cross-linking studies reveal orientation of tRNA in the ribosomeModel of 70S ribosomal complex with A, P, and E sites occupied by tRNAsProtein synthesis IBiochemistry 302Bob Kelm February 23, 2004Key features of protein synthesis• Energy glutton– Essential metabolic activity of the cell.– Consumes ∼90% of the chemical energy (ATP,GTP).– Components of translational machinery account for ∼35% of the dry weight of the cell.• Fast and Accurate– Polypeptide of 100 amino acids synthesized in ∼ 5 sec.– Error rate of 1 in 10,000 to 50,000.• Highly regulated– Coordination of rRNA and protein synthesis– Ribosome activity/assemblyPrimary steps involved in synthesizing a functional protein• Activation of amino acids– Joining amino acids to their cognate tRNA• Initiation of protein synthesis– Assembling the ribosome on the mRNA• Elongation of polypeptide chain – Creating peptide bonds between amino acids• Termination of translation– Completing the polypeptide chain and releasing ribosomes• Folding and Processing– Covalent modification of certain amino acidsSubunit composition of the prokaryotic ribosome (~2/3 rRNA, 1/3 protein)E. coli ribosome:∼15,000/cell∼ 25% dry wt70S → 2.3 MDaS1-S211542 nt2904 nt120 ntL1-L31Fig. 27.13S = M(1-νρ)/NfL1-L31 and S1-21 are small, non-homologous proteins that are highly conserved from organism to organism.Ribosomal components: prokaryotes versus eukaryotes Prokaryotic 70S Eukaryotic 80SLarge Subunit 50S 60S RNA 23S rRNA (2.9 kb) 28S rRNA (4.7 kb) 5S rRNA (120 nt) 5S rRNA (120 nt) 5.8S rRNA Proteins 31 (L1,L2,L3…) 49 Small subunit 30S 40S RNA 16S rRNA (1.5 kb) 18S rRNA (1.8 kb)Proteins 21 (S1,S2,S3…) 33Assembly of small ribosomal subunit is an ordered process in vitroReconstitution of 50S subunit proceeds by a more complex pathway that requires careful temperature control.Fig. 27.19Reconstitution of 30S subunits from individual components 1streported by P. Traub and M. Nomura in 1968.Monovalent & divalent cations modulate 70S ribosome assembly in vitro30S + 50S 70S↑[Mg2+]30S + 50S 70S↑[Na+/K+]Under the ionic conditions present in the cell, ribosomes exist primarily as dissociated subunits.Ribosomal subunits have distinct function roles in protein synthesis• Small subunit (recognition & specificity)– Initiates mRNA engagement– Decodes the mRNA – Mediates mRNA and tRNA translocation– Ensures high fidelity codon-anticodon interaction• Large subunit (catalysis & regulation)– Catalyzes peptide bond formation– Provides a route for nascent peptide growth (tunnel)– Provides binding sites for G protein factors that assist in initiation, elongation, and termination phases of protein synthesisPutative secondary structure of E. coli16S rRNA• Many regions of self-complementarity facilitate intra-strand base pairing revealing four major domains of folding (I-IV).• Predicted double-stranded regions are highly conservedamong related 16S rRNA sequences but primary sequences are not.• Additional folding of rRNA and contribution of ribosomal proteins generate a more realistic 3D structure.Fig. 27.155′3′Early EM studies reveal 3D topography of large and small ribosomal subunits30S 50S 70SFig. 27.16Side viewFront view3D-structure of small ribosomal subunit of Thermus thermophilus(shape determined by RNA component)Note asymmetric arrangement of proteins and RNA. IIIIVfront view: 50S interaction surfacemRNA pathIIIB. T. Wimberly et al. Nature 407:327-339, 2000, 3.3 angstrom resolutionH:head, Be:Beak, N:neck, P:platform, Sh:shoulder, Sp:Spur, Bo:body3D-structure of small ribosomal subunit of Thermus thermophilus (decoding center made entirely of RNA)Aperture of a “latch”NoseGateway to de-coding centerF. Schluenzen et al. Cell 102:615-623, 2000 from Max-Planck Institute, 3.3 angstrom resolution, 1 angstrom = 10-10mNot to be outdone…Steitz group determines crystal structure of 50S ribosomal subunit of Haloarcula marismortuiPeptidyl transferase inhibitor Note that proteins are remote from active site, primary role in stabilizing 3D rRNA structure.5S rRNA regionRidgeMonolithic structure with two lateral protuberances The surface of the subunit that interacts with the small 30S subunit faces you.N. Ban et al. Science 289:905-920, 2000 2.4 angstrom resolutionA representative prokaryotic mRNA: the lac operon (a polycistronic message)Fig. 27.5Signals for ribosome binding and translation initiation, some better (↑affinity) than othersInitiation requires ribosomal alignment:Shine-Dalgarno sequencesSD sequences: Purine-rich sequences that function as attachment sites for 3′ end of 16S rRNA.Table 27.3Essential prokaryotic protein factors involved in translationTable 27.4**Initiation of protein synthesis in prokaryotes• IF1 and IF3 bind 30S subunit kicking off 50S.• IF1:IF3:30S subunit complex simultaneously acquires IF2:GTP:fMet-tRNA complex and mRNA. Shine-Dalgarno sequence mediates 30S and IF2:GTP:fMet-tRNAfMetbinding. IF2 is a G protein (GTPase).• 50S subunit joins the 30S preinitiation complex. IF1 and IF3 are released. GTP is hydrolyzed with release of IF2 and GDP. 70S initiation complex is ready for 2ndcharged tRNA and elongation. E=exit P=peptidyl A=aminoacyl


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UVM BIOC 302 - Protein synthesis I

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