Initiation Factors in the Early Events of mRNA Translation in Bacteria

Gualerzi, Claudio O.; Brandi, Letizia; Caserta, Enrico; Garofalo, Cristiana; Lammi, M.; Teana, Anna La; Petrelli, Dezemona; Spurio, Roberto; Tomšič, Jerneja; Pon, Cynthia L.

doi:10.1101/sqb.2001.66.363

Cited by 90 publications

(118 citation statements)

References 78 publications

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“…60 and 61, reviewed in Ref. 62), an effect similar to that observed when the S7-S11 interaction is disrupted. More recently, cryo-EM studies showed that the regions surrounding the cleft between the head and the platform moves upon binding of IF3 (58).…”

Section: Discussionmentioning

confidence: 64%

A Functional Interaction between Ribosomal Proteins S7 and S11 within the Bacterial Ribosome

Robert¹,

Brakier‐Gingras

2003

Journal of Biological Chemistry

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In this study, we used site-directed mutagenesis to disrupt an interaction that had been detected between ribosomal proteins S7 and S11 in the crystal structure of the bacterial 30 S subunit. This interaction, which is located in the E site, connects the head of the 30 S subunit to the platform and is involved in the formation of the exit channel through which passes the 30 S-bound messenger RNA. Neither mutations in S7 nor mutations in S11 prevented the incorporation of the proteins into the 30 S subunits but they perturbed the function of the ribosome. In vivo assays showed that ribosomes with either mutated S7 or S11 were altered in the control of translational fidelity, having an increased capacity for frameshifting, readthrough of a nonsense codon and codon misreading. Toeprinting and filter-binding assays showed that 30 S subunits with either mutated S7 or S11 have an enhanced capacity to bind mRNA. The effects of the S7 and S11 mutations can be related to an increased flexibility of the head of the 30 S, to an opening of the mRNA exit channel and to a perturbation of the proposed allosteric coupling between the A and E sites. Altogether, our results demonstrate that S7 and S11 interact in a functional manner and support the notion that protein-protein interactions contribute to the dynamics of the ribosome.The ribosome is the cellular machinery responsible for protein synthesis in all living organisms. The elucidation of the crystal structure of the prokaryotic ribosome has led to a major progress in understanding its function (1-7). Analysis of the ribosome structure combined with a wealth of biochemical data clearly showed that ribosomal RNA (rRNA) is the key player in the functions of the ribosome, and it is currently assumed that the main task of the ribosomal proteins is to help and stabilize rRNA folding and to facilitate conformational changes in rRNA (8 -10). However, a growing list of examples suggests that ribosomal proteins directly participate to protein synthesis. The x-ray structure of the ribosome reveals that S12 is part of the decoding site (Refs. 11 and 12, reviewed in Ref. 13). It also shows that the 30 S proteins S13, S15, and the 50S proteins L2, L5, L14, L19 are involved in intersubunit bridges, while the 30 S proteins S9, S13, and the 50 S proteins L1, L5, L33 interact with tRNAs, suggesting that they could participate in the translocation step (12). Studies in solution also point to a role for the ribosomal proteins, such as an involvement in mRNA binding for S1 (14 -16), a participation in peptidyl transferase activity for L2 (17-19) and the prevention of mRNA slippage for L9 (20).Several protein-protein interactions were identified in the crystal structure of the 30 S subunit (21, 22) but, so far, only the interaction between proteins S4 and S5 has been directly related to a particular ribosome function, that is the control of translational fidelity. Indeed, mutations that disrupt the S4-S5 interaction decrease translational accuracy (23,24). Among the other protein-protein inter...

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Section: Discussionmentioning

confidence: 64%

A Functional Interaction between Ribosomal Proteins S7 and S11 within the Bacterial Ribosome

Robert¹,

Brakier‐Gingras

2003

Journal of Biological Chemistry

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“…These studies reveal that the SD helix sits in a pocket located between the head and platform of the 30S subunit, adjacent to but not directly in the E-site. The SD-anti-SD interaction probably reduces the time necessary for mRNA-ribosome programming, because it helps to guide the mRNA from an initial stand-by site into a position whereby the AUG start codon is correctly positioned in the presence of initiator tRNA (36,38,39). Interestingly, the conformation of the mRNA in the E-site appears to be influenced by the state of the ribosome.…”

Section: Discussionmentioning

confidence: 99%

Shine–Dalgarno interaction prevents incorporation of noncognate amino acids at the codon following the AUG

Giacco

Márquez

Qin

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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During translation, usually only one in Ϸ400 misincorporations affects the function of a nascent protein, because only chemically similar near-cognate amino acids are misincorporated in place of the cognate one. The deleterious misincorporation of a chemically dissimilar noncognate amino acid during the selection process is precluded by the presence of a tRNA at the ribosomal E-site. However, the selection of first aminoacyl-tRNA, directly after initiation, occurs without an occupied E-site, i.e., when only the P-site is filled with the initiator tRNA and thus should be highly error-prone. Here, we show how bacterial ribosomes have solved this accuracy problem: In the absence of a Shine-Dalgarno (SD) sequence, the first decoding step at the A-site after initiation is extremely error-prone, even resulting in the significant incorporation of noncognate amino acids. In contrast, when a SD sequence is present, the incorporation of noncognate amino acids is not observed. This is precisely the effect that the presence of a cognate tRNA at the E-site has during the elongation phase. These findings suggest that during the initiation phase, the SD interaction functionally compensates for the lack of codon-anticodon interaction at the E-site by reducing the misincorporation of near-cognate amino acids and prevents noncognate misincorporation. E-site ͉ translational errorsT he binding of aminoacyl-tRNAs to the ribosome is dictated by the complementarity between the anticodon of the tRNA and the codon of the mRNA. To ensure the high fidelity of translation, the correct stereochemistry of the mRNA-tRNA codon-anticodon interaction is monitored by components of the small ribosomal subunit in a process known as decoding (reviewed in ref. 1). During decoding, the first and second nucleotide positions (in terms of the codon) of the mRNA-tRNA duplex are closely monitored, whereas interaction at the third or wobble position is less strictly recognized. Consistently, the misincorporation of the wrong amino acids into polypeptide chains usually occurs through the binding of nearcognate aminoacyl-tRNAs, i.e., those tRNAs carrying an anticodon similar to that of the cognate aminoacyl-tRNA, rather than noncognate aminoacyl-tRNAs, which carry dissimilar anticodons. Nascent polypeptide chains are surprisingly tolerant to misincorporation, with only one in Ϸ400 misincorporations being deleterious for the protein's activity (reviewed in ref.2). The reason for this is that usually near-cognate aminoacyl-tRNAs are selected instead of the cognate aminoacyl-tRNA, and the genetic code lexicon is organized in such a way that near-cognate tRNAs bear amino acids that are chemically similar to those carried by the cognate tRNA. For example, the misincorporation of an aspartate (codon: GAU/C) by near-cognate Asp-tRNA, instead of glutamate (GAA/G) by the cognate Glu-tRNA, both incorporate acidic amino acids. The middle and, in most cases, the first position of a codon are almost never misread, even under error-inducing conditions such as high magnesiu...

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“…Both the fidelity and speed of 70SIC formation require the presence of initiation factors IF1, IF2, and IF3. The guanine nucleotide binding protein IF2 plays a crucial role, interacting directly with fMet-tRNA fMet , favoring its decoding in the P-site and physically linking the 30S and 50S subunits (for reviews see references [1][2][3][4][5].…”

Section: Introductionmentioning

confidence: 99%

A Quantitative Kinetic Scheme for 70 S Translation Initiation Complex Formation

Grigoriadou

Marzi

Kirillov

et al. 2007

Journal of Molecular Biology

196

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SUMMARYAssociation of the 30S initiation complex (30SIC) and the 50S ribosomal subunit, leading to formation of the 70S initiation complex (70SIC), is a critical step of the translation initiation pathway. The 70SIC contains initiator tRNA, fMet-tRNA fMet , bound in the P (peptidyl)-site in response to the AUG start codon. We have formulated a quantitative kinetic scheme for the formation of an active 70SIC from 30SIC and 50S subunits on the basis of parallel rapid kinetics measurements of GTP hydrolysis, Pi release, light scattering, and changes in fluorescence intensities of fluorophore-labeled IF2 and . According to this scheme, an initially formed labile 70S complex, which promotes rapid IF2-dependent GTP hydrolysis, either dissociates reversibly into 30S and 50S subunits or is converted to a more stable form, leading to 70SIC formation. The latter process takes place with intervening conformational changes of ribosome-bound IF2 and fMet-tRNA fMet , which are monitored by spectral changes of fluorescent derivatives of IF2 and fMet-tRNA fMet . The availability of such a scheme provides a useful framework for precisely elucidating the mechanisms by which substituting the nonhydrolyzable analogue GDPCP for GTP or adding thiostrepton inhibit formation of a productive 70SIC. GDPCP does not affect stable 70S formation, but perturbs fMettRNA fMet positioning in the P-site. In contrast, thiostrepton severely retards stable 70S formation, but allows normal binding of fMet-tRNA fMet (prf20) to the P-site.

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Initiation Factors in the Early Events of mRNA Translation in Bacteria

Cited by 90 publications

References 78 publications

A Functional Interaction between Ribosomal Proteins S7 and S11 within the Bacterial Ribosome

A Functional Interaction between Ribosomal Proteins S7 and S11 within the Bacterial Ribosome

Shine–Dalgarno interaction prevents incorporation of noncognate amino acids at the codon following the AUG

A Quantitative Kinetic Scheme for 70 S Translation Initiation Complex Formation

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