Initiation of protein synthesis from a termination codon.
Prior to peptide bond formation, an aminoacyl-tRNA is bound in the ribosomal A-site, a peptidyl-tRNA is bound in the P-site, and a deacylated tRNA, which is ready for ejection from the ribosome, is bound to the E-site. Translation moves the tRNA from the A-site through the P- and E-sites before they exit the ribosome again, with the exception of the initiator tRNA, which binds directly to the P-site. The small ribosomal subunit contains the decoding center, where the triplet codons of the mRNA are base-paired with the anticodons of the cognate tRNA, and hence determines the sequence of amino acids to be incorporated in the synthesized protein. The large subunit contains the peptidyltransferase center and is thus the catalytic unit.
TRANSLATION INITIATION (Protein Synthesis) - what …
Two genes in the E. coli genome encode tRNAfMet (). The major fraction of cellular initiator tRNA (tRNAf1Met) is encoded by the metZ gene. Three identical copies of the gene occur in tandem repeats within the operon known as the metZ operon (). A relatively small fraction of tRNAfMet (tRNAf2Met) is encoded by the metY gene, located at the beginning of the nusA/infB operon (). The presence of adenosine instead of 7-methylguanosine at position 46 is the only difference between tRNAf2Met and tRNAf1Met ().
Methionine-isoaccepting initiator and elongator tRNAs are both aminoacylated by methionyl tRNA synthetase. Two identical subunits form the dimeric native enzyme, which binds two tRNAMet molecules in an anticooperative manner (, ). MetRS interacts with part of the acceptor stem and the anticodon loop of the tRNA (Fig. ). The major determinant for MetRS in tRNAMet binding is the anticodon. Aminoacylation with methionine is not possible if this triplet is mutated, whereas other tRNAs provided with a CAU anticodon can be methionylated by MetRS. Recognition of the anticodon by MetRS occurs through the helical C-terminal region of the synthetase ().
Initiation of Protein Synthesis in Escherichia coli, I
E. coli IF3 is a 20.4-kDa protein composed of 180 amino acids encoded by the essential infC gene (, ). The infC-rpmI-rplT operon contains the genes encoding IF3 and the two ribosomal proteins L35 and L20 (, ). These genes are transcribed from four promoters and terminated by two transcriptional terminators (, ). At the translational level, the expression from the operon is regulated by two different control circuits, discussed further in the last section of this review. Whereas IF1 and IF2 are universally present and important for the function of all living cells, IF3 is limited to a number of bacterial species and has been found in some plastids (, , ). The human mitochondrial IF3mt has short extensions in the N and C termini surrounding a region homologous to bacterial IF3. It promotes initiation complex formation on mitochondrial ribosomes ().
Biology - Ribosomes and Protein Synthesis
IF2 has been cross-linked to S13, L7-L12, IF1, and IF3 (), as well as S1, S2, S11, S12, and S19 on the ribosome (). Chemical probing experiments with 23S rRNA indicated that IF2 protects A2476 and A2478 in helix 89 of domain V as well as G2655, A2660, G2661, and A2665 of the sarcin-ricin domain positioned in domain VI (Fig. ) (). These footprints were generated regardless of the presence of GTP, IF1, mRNA, and fMet-tRNAfMet. Unfortunately, the results are unclear with respect to the 30S ribosomal subunit, since IF2 affects the reactivity of residues spread all over the subunit. This is consistent with an observed rearrangement of the subunit induced by IF2 (). Recently, a model of IF2 binding to the ribosome was presented, based on cleavage of the rRNA by chemical nucleases tethered to cysteine residues introduced at specific sites of IF2 (). No cleavage of the 16S rRNA was observed when IF2 was bound to 30S ribosomal subunits or to the complete 30S initiation complex. However, cleavage of the 16S rRNA was observed when IF2 was bound to the 70S initiation complex (Fig. ). These data indicate that domain V of IF2 is localized toward the 30S subunit in the 70S initiation complex. As described above, cross-linking data of the 30S complex and footprinting data on the binary fMet-tRNAfMet-IF2 complex place domain V of IF2 in proximity to the elbow of the P-site-bound fMet-tRNAfMet. The distance between the 16S rRNA and the elbow of the fMet-tRNAfMet appears to be too far for domain V of IF2 to establish contact with both simultaneously. Conclusively, IF2 changes localization during the transition from the 30S to the 70S initiation complex (). The cleavage experiments were performed in the presence of excess GTP. Large domain movements take place in IF2 during GTP hydrolysis (), and the cleavage patterns in the rRNA might be dependent on whether IF2 is in the GTP- or GDP-bound form. To fully understand the function of IF2 during translation initiation, detailed atomic resolution structures of both the 30S and 70S initiation complexes as well as a better understanding of the timing and not least the consequences of GTP hydrolysis are needed.
Protein synthesis begins with the formation of an initiation complex
As mentioned above, the interactions between fMet-tRNAfMet and IF2 have been studied extensively. Formation of the binary complex is strongly dependent on the formylation of Met-tRNAfMet () but independent of GTP (). The C-terminal domain VI-2 of IF2 has been suggested to contain the entire binding site for fMet-tRNAfMet (). The interaction of the domain with fMet-tRNAfMet has been studied using mutagenesis as well as Raman and NMR spectroscopy (, , , ). Functionally essential residues of the B. stearothermophilus domain are C668, K699, R700, Y701, K702, E713, C714, and G715 (Fig. ). Cross-linking experiments indicate interactions between E. coli residues N611-R645 (belonging to domain V) and the T-stem of fMet-tRNAfMet as well as residues W215-R237 (domain II) and the anticodon stem of fMet-tRNAfMet (, ). Finally, fMet-tRNAfMet protects a position in domain IV and weakly in domain V of B. stearothermophilus IF2 against digestion by trypsin (). A stable interaction between the archaeal and eukaryotic IF2 homologues and Met-tRNAiMet has not been observed in vitro, but overexpression of the gene encoding tRNAiMet partially suppresses the severe slow-growth phenotype of yeast strains lacking the IF2 homologue ().