Protein synthesis I Biochemistry 302. Bob Kelm February 23, 2004

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1 Protein synthesis I Biochemistry 302 Bob Kelm February 23, 2004

2 Key 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/assembly

3 Primary 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 acids

4 Subunit composition of the prokaryotic ribosome (~2/3 rrna, 1/3 protein) E. coli ribosome: 15,000/cell L1-L31 25% dry wt 70S 2.3 MDa S = M(1-νρ)/Nf 2904 nt S1-S nt Fig nt L1-L31 and S1-21 are small, non-homologous proteins that are highly conserved from organism to organism.

5 Ribosomal components: prokaryotes versus eukaryotes Prokaryotic 70S Large Subunit 50S 60S RNA Eukaryotic 80S 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 ) 33

6 Assembly of small ribosomal subunit is an ordered process in vitro Reconstitution of 30S subunits from individual components 1 st reported by P. Traub and M. Nomura in Fig Reconstitution of 50S subunit proceeds by a more complex pathway that requires careful temperature control.

7 Monovalent & divalent cations modulate 70S ribosome assembly in vitro 30S + 50S [Mg 2+ ] 70S 30S + 50S [Na + /K + ] 70S Under the ionic conditions present in the cell, ribosomes exist primarily as dissociated subunits.

8 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 synthesis

9 Putative secondary structure of E. coli 16S rrna Many regions of selfcomplementarity facilitate intra-strand base pairing revealing four major domains of folding (I-IV). Predicted double-stranded regions are highly conserved among related 16S rrna sequences but primary sequences are not. Additional folding of rrna and contribution of ribosomal proteins generate a more realistic 3D structure. 5 3 Fig

10 30S 50S 70S Early EM studies reveal 3D topography of large and small ribosomal subunits Fig Side view Front view

11 H:head, Be:Beak, N:neck, P:platform, Sh:shoulder, Sp:Spur, Bo:body B. T. Wimberly et al. Nature 407: , 2000, 3.3 angstrom resolution 3D-structure of small ribosomal subunit of Thermus thermophilus (shape determined by RNA component) mrna path II III front view: 50S interaction surface I IV Note asymmetric arrangement of proteins and RNA.

12 3D-structure of small ribosomal subunit of Thermus thermophilus (decoding center made entirely of RNA) Aperture of a latch Gateway to decoding center Nose F. Schluenzen et al. Cell 102: , 2000 from Max-Planck Institute, 3.3 angstrom resolution, 1 angstrom = m

13 Not to be outdone Steitz group determines crystal structure of 50S ribosomal subunit of Haloarcula marismortui Peptidyl transferase inhibitor Note that proteins are remote from active site, primary role in stabilizing 3D rrna structure. Ridge 5S rrna region Monolithic 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: , angstrom resolution

14 A representative prokaryotic mrna: the lac operon (a polycistronic message) Fig Signals for ribosome binding and translation initiation, some better ( affinity) than others

15 Initiation requires ribosomal alignment: Shine-Dalgarno sequences Table 27.3 SD sequences: Purine-rich sequences that function as attachment sites for 3 end of 16S rrna.

16 Essential prokaryotic protein factors involved in translation Table 27.4 * *

17 Fig 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-tRNA fmet binding. 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 2 nd charged trna and elongation. E=exit P=peptidyl A=aminoacyl binding sites Initiator trna is special because it carries fmet.

18 A word about the prokaryotic initiator trna, fmet-trna fmet Formyl group is added after charging of trna fmet with Met. Transformylase catalyzes the transfer of a formyl group from N 10 -formylthf to Met-tRNA fmet. All prokaryotic mrnas are synthesized with the same N-terminal residue, N-fMet. Formyl group is removed during chain elongation by deformylase. Fig. 5.17

19 Chemical cross-linking studies reveal orientation of trna in the ribosome A site, triangles P site, circles E site, squares Acceptor end interacts with 50S subunit near the top of the 70S ribosome cavity. Fig Anticodon end contacts 30S subunit near bottom of 70S ribosome cavity.

20 Model of 70S ribosomal complex with A, P, and E sites occupied by trnas 70S cavity Fig S tunnel View with 30S subunit in front, 50S subunit behind These models show all three sites (A, P, E) occupied by trnas. This would never occur during protein synthesis.