Conserved residues in yeast initiator tRNA calibrate initiation accuracy by regulating preinitiation complex stability at the start codon

Self Cite

The eukaryotic 43S preinitiation complex (PIC) bearing Met-tRNA i Met in a ternary complex (TC) with eukaryotic initiation factor (eIF)2-GTP scans the mRNA leader for an AUG codon in favorable "Kozak" context. AUG recognition provokes rearrangement from an open PIC conformation with TC bound in a state not fully engaged with the P site ("P OUT ") to a closed, arrested conformation with TC tightly bound in the "P IN " state. Yeast ribosomal protein Rps3/uS3 resides in the mRNA entry channel of the 40S subunit and contacts mRNA via conserved residues whose functional importance was unknown. We show that substitutions of these residues reduce bulk translation initiation and diminish initiation at near-cognate UUG start codons in yeast mutants in which UUG selection is abnormally high. Two such substitutions-R116D and R117D-also increase discrimination against an AUG codon in suboptimal Kozak context. Consistently, the Arg116 and Arg117 substitutions destabilize TC binding to 48S PICs reconstituted in vitro with mRNA harboring a UUG start codon, indicating destabilization of the closed P IN state with a UUGanticodon mismatch. Using model mRNAs lacking contacts with either the mRNA entry or exit channels of the 40S subunit, we demonstrate that Arg116/Arg117 are crucial for stabilizing PIC-mRNA contacts at the entry channel, augmenting the function of eIF3 at both entry and exit channels. The corresponding residues in bacterial uS3 promote the helicase activity of the elongating ribosome, suggesting that uS3 contacts with mRNA enhance multiple phases of translation across different domains of life.ccurate identification of the translation initiation codon in mRNA by ribosomes is crucial for expression of the correct cellular proteins. This process generally occurs in eukaryotic cells by a scanning mechanism wherein the small (40S) ribosomal subunit first recruits charged initiator tRNA (Met-tRNA i Met ) in a ternary complex (TC) with eukaryotic initiation factor (eIF)2-GTP in a reaction stimulated by eIFs 1, 1A, 3, and 5. The resulting 43S preinitiation complex (PIC) attaches to the 5′ end of the mRNA and scans the 5′ UTR with TC bound in a metastable state, "P OUT ," suitable for inspecting successive triplets for complementarity with the anticodon of Met-tRNA i Met in the P site, to identify the AUG start codon. Nucleotides surrounding the AUG, particularly at the −3 and +4 positions (the Kozak context), further influence the efficiency of start-codon selection. In the scanning PIC, eIF2 can hydrolyze GTP, dependent on GTPase-activating protein eIF5, but P i release is blocked by eIF1, whose presence also impedes stable binding of Met-tRNA i Met in the "P IN " state. Start-codon recognition triggers dissociation of eIF1 from the 40S subunit, allowing P i release from eIF2-GDP•P i and TC binding in the P IN state of the 48S PIC (Fig. 1A). Subsequent dissociation of eIF2-GDP and other eIFs from the 48S PIC enables eIF5B-catalyzed subunit joining and formation of an 80S initiation complex with Met-tRNA i Met basepaired...

Section: Rps3supporting

confidence: 90%

Section: Rps3mentioning

confidence: 88%

Rps3/uS3 promotes mRNA binding at the 40S ribosome entry channel and stabilizes preinitiation complexes at start codons

Dong

Aitken

Thakur

et al. 2017

Self Cite

“…8A). In yeast, the 3GC tRNAs also showed stable binding to the 40S (28,29) but not the 80S ribosomes (28), supporting our proposal of the role of the 3GC pairs, a feature of initiator tRNAs conserved in all domains of life. In vitro studies using 70S ribosomes also showed that the 3GC pairs are required for tRNA fMet binding to the ribosome (4).…”

Section: Discussionsupporting

confidence: 69%

An extended Shine–Dalgarno sequence in mRNA functionally bypasses a vital defect in initiator tRNA

Shetty

Nadimpalli

Shah

et al. 2014

Initiator tRNAs are special in their direct binding to the ribosomal P-site due to the hallmark occurrence of the three consecutive G-C base pairs (3GC pairs) in their anticodon stems. How the 3GC pairs function in this role, has remained unsolved. We show that mutations in either the mRNA or 16S rRNA leading to extended interaction between the Shine-Dalgarno (SD) and anti-SD sequences compensate for the vital need of the 3GC pairs in tRNA fMet for its function in Escherichia coli. In vivo, the 3GC mutant tRNA fMet occurred less abundantly in 70S ribosomes but normally on 30S subunits. However, the extended SD:anti-SD interaction increased its occurrence in 70S ribosomes. We propose that the 3GC pairs play a critical role in tRNA fMet retention in ribosome during the conformational changes that mark the transition of 30S preinitiation complex into elongation competent 70S complex. Furthermore, treating cells with kasugamycin, decreasing ribosome recycling factor (RRF) activity or increasing initiation factor 2 (IF2) levels enhanced initiation with the 3GC mutant tRNA fMet , suggesting that the 70S mode of initiation is less dependent on the 3GC pairs in tRNA fMet . 3GC base pairsnitiation of protein synthesis, assisted by initiation factors, is a highly regulated process in all life forms. In eubacteria, binding of both the initiator tRNA (tRNA fMet ) and mRNA to the small ribosomal subunit (30S) leads to the formation of a 30S preinitiation complex primarily with the help of the three initiation factors (IF1, IF2, and IF3). This stage is then followed by docking of the large ribosomal subunit (50S) to ultimately produce an elongation competent 70S complex upon the departure of all of the three initiation factors (1). The localization of mRNA onto the 30S subunit is facilitated by a purine rich sequence (Shine-Dalgarno, SD sequence), located upstream of the start codon, by its pairing with a complementary sequence (anti-SD sequence) at the 3′-terminus of the 16S rRNA (1, 2). The tRNA fMet binding to ribosome is aided by the unique features it possesses. A virtually universal feature of all of the initiator tRNAs, the presence of three consecutive G-C base pairs (G 29 G 30 G 31 :C 39 C 40 C 41 , referred to as 3GC pairs) in the anticodon stem is known to be important for their preferential binding in the ribosomal P-site (3, 4). Mutations in the 3GC pairs result in poor binding of tRNA fMet to the ribosomal P-site (3, 4).However, the mechanism of how the 3GC pairs help in binding of tRNA fMet into the ribosome has remained unclear. In the crystal structure of the initiator tRNA bound 70S ribosome, it was seen that the universally conserved A1339 and G1338 residues of 16S rRNA establish A-minor interactions with the first GC (G 29 -C 41 ) and middle GC (G 30 -C 40 ) pairs, respectively (5). Although these interactions were seen as suboptimal, the IF3 induced conformational changes may optimize these interactions (6). Another study showed that a major role of IF3 is to uniformly increase the rate of dissociation of...

“…4G). Previous work indicated that TC bound in the P OUT conformation is too unstable to remain associated with the PIC during native gel electrophoresis, such that the measured rate of TC dissociation in these assays largely reflects the proportion of complexes in the P IN state and the stability of that state (22,23). In agreement with previous findings (22), we observed that TC dissociates more rapidly from the mRNA(UUG) versus mRNA(AUG) PICs in reactions containing WT eIF1 ( Fig.…”

Section: Substitutions Of Eif1 Loop 2 Residues Decrease Discriminationmentioning

confidence: 99%

eIF1 Loop 2 interactions with Met-tRNA _i control the accuracy of start codon selection by the scanning preinitiation complex

Thakur

Hinnebusch

2018

Self Cite

The eukaryotic 43S preinitiation complex (PIC), bearing initiator methionyl transfer RNA (Met-tRNA) in a ternary complex (TC) with eukaryotic initiation factor 2 (eIF2)-GTP, scans the mRNA leader for an AUG codon in favorable context. AUG recognition evokes rearrangement from an open PIC conformation with TC in a "P" state to a closed conformation with TC more tightly bound in a "P" state. eIF1 binds to the 40S subunit and exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the P state, necessitating eIF1 dissociation for start codon selection. Structures of reconstituted PICs reveal juxtaposition of eIF1 Loop 2 with the Met-tRNA D loop in the P state and predict a distortion of Loop 2 from its conformation in the open complex to avoid a clash with Met-tRNA We show that Ala substitutions in Loop 2 increase initiation at both near-cognate UUG codons and AUG codons in poor context. Consistently, the D71A-M74A double substitution stabilizes TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, without affecting eIF1 affinity for 40S subunits. Relatively stronger effects were conferred by arginine substitutions; and no Loop 2 substitutions perturbed the rate of TC loading on scanning 40S subunits in vivo. Thus, Loop 2-D loop interactions specifically impede Met-tRNA accommodation in the P state without influencing the P mode of TC binding; and Arg substitutions convert the Loop 2-tRNA clash to an electrostatic attraction that stabilizes P and enhances selection of poor start codons in vivo.