Selection of the initiator tRNA by Escherichia coli initiation factors.

Ribosome crystal structures have revealed that two small subunit proteins, S9 and S13, have C-terminal tails, which, together with several features of 16S rRNA, contact the anticodon stem-loop of P-site tRNA. To test the functional importance of these protein tails, we created genomic deletions of the C-terminal regions of S9 and S13. All of the tail deletions, including double mutants containing deletions in both S9 and S13, were viable, showing that Escherichia coli cells can synthesize all of their proteins by using ribosomes that contain 30S P sites composed only of RNA. However, these mutants have slower growth rates, indicating that the tails may play a supporting functional role in translation. In vitro analysis shows that 30S subunits purified from the S13 deletion mutants have a generally decreased affinity for tRNA, whereas deletion of the S9 tail selectively affects the binding of tRNAs whose anticodon stem sequences are most divergent from that of initiator tRNA. E xtensive evidence from more than three decades of experiments (1) supports the idea that ribosomal function is based primarily on rRNA (2, 3). This is most clearly seen in ribosome crystal structures, which show that the functional region of the ribosome, at the subunit interface, is composed mostly of rRNA, whereas the ribosomal proteins are found mainly at the periphery of the ribosome (4-7). However, there are a number of instances where ribosomal proteins contact tRNA (6). One of these occurs in the 30S P site, which plays a major role in initiation of protein synthesis, binding the initiator tRNA and positioning the start codon of mRNA. The 30S P site is also responsible for binding and positioning the anticodon stem-loop (ASL) of peptidyl-tRNA during polypeptide elongation and for maintaining the translational reading frame when the A site is vacant. Binding of the ASL depends not only on base-pairing with the P-site codon of mRNA, but also on interactions with the 30S subunit itself. In addition to contacts involving several elements of 16S rRNA, the extended C-terminal tails of two proteins, S9 and S13, penetrate the 30S P site within contact distance of the ASL (5, 6). The universally conserved C-terminal arginine of S9 appears to contact phosphate 35 at the apex of the anticodon loop of the P-site tRNA (Fig. 1). The C-terminal tail of S9 is phylogenetically invariant in length, an observation that is explained by the crystal structure, which shows that the P-site tRNA would physically block extension of the S9 tail. The tail of S13 runs parallel to the anticodon stem, crossing the tRNA backbone between positions 29 and 30 and is thus more tolerant of variations in length (Fig. 1). Although more variable in sequence, the S13 tail always contains several basic side chains, which can make electrostatic interactions with tRNA phosphates. The interactions of the S9 and S13 protein tails with P-site tRNA and their phylogenetic conservation suggest that they are directly involved in the function of the 30S P site.Early in vitro reconsti...

Section: Resultsmentioning

confidence: 99%

Creating ribosomes with an all-RNA 30S subunit P site

Hoang

Fredrick

Noller

2004

“…The first function of IF3 is related to the accuracy of initiation, because IF3 increases the dissociation rate of noncanonical 30SIC (56), a function that is restricted to 30SIC rather than to 70SIC (17), leaving IF3's important role for 70S scanning as its main second function.…”

Section: Discussionmentioning

confidence: 99%

“…All three factors are thought to dissociate upon 50S arrival or shortly thereafter (1). IF1 is required for proper initiator-tRNA selection on 70S along with IF2 and IF3, in contrast to the 30SIC, where IF2 and IF3 provide tRNA selection (17).…”

mentioning

confidence: 99%

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

Yamamoto

Wittek

Gupta

et al. 2016

106

According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1-IF3). Here, we describe a novel type of initiation termed "70S-scanning initiation," where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine-Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.protein synthesis | ribosomal functions | translational initiation | 30S-binding initiation | 70S-scanning initiation I t is textbook knowledge that 30S subunits initiate protein synthesis in bacteria; they recognize the initiation site of the mRNA composed of the Shine-Dalgarno (SD) sequence, the AUG codon, and fMet-tRNA, together with three initiation factors (IFs) forming the 30S initiation complex (30SIC). Association of the large 50S subunit triggers the release of the IFs, leading to the 70S initiation complex (70SIC) that enters the elongation phase of translation (reviewed in 1). We term this initiation path the "30S-binding mode" of bacterial initiation. After elongation and termination, it is thought that the ribosome dissociates into its subunits, thus providing 30S subunits for the next round of initiation.The functional role of IF2 is well defined. It can bind directly to the 30S, providing a docking site for fMet-tRNA (2), but it can also enter the 30S subunit as ternary complex fMet-tRNA•IF2•GTP (3). Both IF2 and IF3 are essential for viability. IF3 has a binding site at the 30S interface (4), which explains its antiassociation effect (5, 6), as well as its role in dissociation of the terminating 70S ribosome (7). However, the in vivo concentration of IF3 is 100-fold less (8) than required for full dissociation of 70S in vitro (4). Evidence for the presence of IF3 on 70S ribosomes was reported (9), indicating that the functional spectrum of IF3 is possibly not restricted to an antiassociation effect. Both IF3 and IF2 are also responsible for the fidelity of decoding the initiation AUG by fMet-tRNA Met f at the P site of 30S subun...

“…Furthermore, the distance from the first nucleotide of the codon-like trinucleotide of PKI (cytosine of the CCU in the TSV IRES, numbered +1) to the mRNA entrance is 12-13 nt, as the toe prints of +13 to +14 report (7,10,17). It is known from the studies on bacterial 70S • tRNA initiation complexes (46,47), as well as on mammalian 80S ribosomes (48) bound with initiator methionyl-tRNA Met and/or IRESs initiating with the canonical AUG codon, including the hepatitis C virus (49) and encephalomyocarditis virus (50) IRESs, that the toe prints of +16 to +17 correspond to the placement of the AUG codon in the P site. The toe prints of +13 to +14, observed for the PSIV, CrPV, and TSV IRESs on eukaryotic ribosomes (7,10,17), are therefore consistent with the placement of the CCU trinucleotide of PKI in the A site in both the 40S and 80S complexes.…”

Section: Tsv Ires Stabilizes Two Partially Rotated Conformations Of Thementioning

confidence: 99%

Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA–like structural element in the ribosomal decoding center

Koh

Brilot

Grigorieff

et al. 2014

116

In cap-dependent translation initiation, the open reading frame (ORF) of mRNA is established by the placement of the AUG start codon and initiator tRNA in the ribosomal peptidyl (P) site. Internal ribosome entry sites (IRESs) promote translation of mRNAs in a capindependent manner. We report two structures of the ribosomebound Taura syndrome virus (TSV) IRES belonging to the family of Dicistroviridae intergenic IRESs. Intersubunit rotational states differ in these structures, suggesting that ribosome dynamics play a role in IRES translocation. Pseudoknot I of the IRES occupies the ribosomal decoding center at the aminoacyl (A) site in a manner resembling that of the tRNA anticodon-mRNA codon. The structures reveal that the TSV IRES initiates translation by a previously unseen mechanism, which is conceptually distinct from initiator tRNA-dependent mechanisms. Specifically, the ORF of the IRES-driven mRNA is established by the placement of the preceding tRNA-mRNA-like structure in the A site, whereas the 40S P site remains unoccupied during this initial step.tRNA-mRNA mimicry | IRES-dependent initiation | factor-independent initiation | FREALIGN | real-space refinement