Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli (original) (raw)
Related papers
Nucleic Acids Research, 1989
For various genes of E.coli, three regions (-55 to-1;-35 to-1;-21 to-1) 5' to AUG codon on mRNA were searched for sites of interaction with colicin fragment of 16S rRNA. The detailed sequence comparison points out that apart from Shine-Dalgarno base pairing, an additional ribosomebinding site, a subsequence of 5'-UGAUCC-3' invariably exists in mRNA for highly expressed genes. Poorly expressed genes appear to be controlled by only Shine-Dalgarno base pairing. The analysis indicates that in the initiator region, the-55 to-1 region contains the signal which decides the efficiency of the translation-initiation. The site on 16S rRNA, 5'-GGAUCA-3' at position 1529, that can base pair to the above site, has a recognition site on 23S rRNA at position 2390. In the light of the conserved nature and accessibility of these sites, it is proposed that the site on 16S rRNA plays a bifunctional role-initially it binds to mRNA from highly expressed genes to form a stable 30S initiation complex, and upon association with 50S subunit it exchanges base pairing with 23S rRNA, thus leaving the site on mRNA free.
Nucleic acids …, 1994
Using a conditional expression system with the temperature-induclble XP L promoter, we previously showed that the single mutations 13U-A and 914A-U, and the double mutation 13U-A and 914A-U In Escherichia coll 16S ribosomal RNA Impair the binding of streptomycin (Pinard et at., The FASEB Journal, 1993, 7,173-176). In this study, we found that the two single mutations and the double mutation increase translational fidelity, reducing In vivo readthrough of nonsense codons and frameshifting, and decreasing In vitro mislncorporatlon In a poly(U)-dlrected system. Using ollgodeoxyribonucleotlde probes which hybridize to the 530 loop and to the 1400 region of 16S rRNA, two regions Involved In the control of tRNA binding to the A site, we observed that the mutations In rRNA Increase the binding of the probe to the 530 loop but not to the 1400 region. We suggest that the mutations at positions 13 and 914 of 16S rRNA induce a conformatlonal rearrangement In the 530 loop, which contributes to the Increased accuracy of the ribosome.
Journal of Bacteriology, 2013
The ribosomal P-site hosts the peptidyl-tRNAs during translation elongation. Which P-site elements support these tRNA species to maintain codon-anticodon interactions has remained unclear. We investigated the effects of P-site features of methylations of G966, C967, and the conserved C-terminal tail sequence of Ser, Lys, and Arg (SKR) of the S9 ribosomal protein in maintenance of the translational reading frame of an mRNA. We generated Escherichia coli strains deleted for the SKR sequence in S9 ribosomal protein, RsmB (which methylates C967), and RsmD (which methylates G966) and used them to translate LacZ from its ؉1 and ؊1 out-of-frame constructs. We show that the S9 SKR tail prevents both the ؉1 and ؊1 frameshifts and plays a general role in holding the P-site tRNA/peptidyl-tRNA in place. In contrast, the G966 and C967 methylations did not make a direct contribution to the maintenance of the translational frame of an mRNA. However, deletion of rsmB in the S9⌬3 background caused significantly increased ؊1 frameshifting at 37°C. Interestingly, the effects of the deficiency of C967 methylation were annulled when the E. coli strain was grown at 30°C, supporting its context-dependent role.
Nucleic Acids Research, 1979
Ultraviolet irradiation (.A = 254 nm) of ternary complexes of E.coli 70 S ribosomes with poly(U) and either Phe-tNtNAPhe (in the A-site) or NAcPhe-tRAPhe (in the P-site) effectively induces covalent linking of tRNA with a limited number of ribosomal proteins. The data obtained indicate that in both sites tRNA is in contact with proteins of both 30 S and 50 S subunits (S5, S7, S9, S10, 2, I6 and L16 proteins in the Asite and S7, S9, SI1, I2, I4, L7/L12 and L27 proteins in the P-site). Similar sets of proteins are in contact with total aminoacyl-tRNA and N-acetylaminoacyl-tRNA. However, here no contacts of tRNA in the P-site with the S7 and L25/S17 proteins were revealed, whereassin the A-site total aminoacyl-tRNA contacts L7/L12. Proteins S9, L2 and, probably, S7 and L7/L12 are common to both sites.
Role of 16S ribosomal RNA methylations in translation initiation in Escherichia coli
The EMBO Journal, 2008
Translation initiation from the ribosomal P-site is the specialty of the initiator tRNAs (tRNA fMet ). Presence of the three consecutive G-C base pairs (G29-C41, G30-C40 and G31-C39) in their anticodon stems, a highly conserved feature of the initiator tRNAs across the three kingdoms of life, has been implicated in their preferential binding to the P-site. How this feature is exploited by ribosomes has remained unclear. Using a genetic screen, we have isolated an Escherichia coli strain, carrying a G122D mutation in folD, which allows initiation with the tRNA fMet containing mutations in one, two or all the three G-C base pairs. The strain shows a severe deficiency of methionine and S-adenosylmethionine, and lacks nucleoside methylations in rRNA. Targeted mutations in the methyltransferase genes have revealed a connection between the rRNA modifications and the fundamental process of the initiator tRNA selection by the ribosome.
Journal of Biological Chemistry, 2006
Ribosomal (r) RNAs play a crucial role in the fundamental structure and function of the ribosome. Helix 69 (H69) (position 1906-1924), a highly conserved stem-loop in domain IV of the 23 S rRNA of bacterial 50 S subunits, is located on the surface for intersubunit association with the 30 S subunit by connecting with helix 44 of 16 S rRNA with the bridge B2a. H69 directly interacts with A/T-, A-, and P-site tRNAs during each translation step. To investigate the functional importance of the highly conserved loop sequence (1912-1918) of H69, we employed a genetic method that we named SSER (systematic selection of functional sequences by enforced replacement). This method allowed us to identify and select from the randomized loop sequences of H69 in Escherichia coli 23 S rRNA functional sequences that are absolutely required for ribosomal function. From a library consisting of 16,384 sequence variations, 13 functional variants were obtained. A1912 and U(⌿)1917 were selected as essential residues in all variants. An E. coli strain having 23 S rRNA with a U to A mutation at position 1915 showed a severe growth phenotype and low translational fidelity. The result could be explained by the fact that the A1915-ribosome variant has weak subunit association, weak A-site tRNA binding, and decreased translational activity. This study proposes that H69 plays an important role in the control of translational fidelity by modulating A-site tRNA binding during the decoding process.
A second putative mRNA binding site on the Escherichia coli ribosome
Gene, 1995
Translation in bacteria is initiated by a base-pairing interaction between the extreme 3'-end of the small-subunit rRNA and a purine-rich domain (Shine-Dalgarno (SD) sequence) preceding the initiation codon at the 5'-end of most bacterial mRNAs. Here, we describe the identification of a second functional and alternative site on the Escherichia coli ribosome which is capable of interacting with mRNA devoid of SD sequences and initiate the translation. This site is localized between nt 1340 and 1360 of the 16S rRNA in E. coli and is complementary to the untranslated region at the 5'-end of tobacco mosaic virus RNA (f~ sequence).
Functional Importance of the 3'-Terminal Adenosine of tRNA in Ribosomal Translation
Journal of Biological Chemistry, 2002
The universally conserved 3-terminal CCA sequence of tRNA interacts with large ribosomal subunit RNA during translation. The functional importance of the interaction between the 3-terminal nucleotide of tRNA and the ribosome was studied in vitro using mutant in vitro transcribed tRNA Val A76G. Val-tRNA CCG does not support polypeptide synthesis on poly(GUA) as a message. However, in a co-translation system, where Val-tRNA CCG represented only a small fraction of total Val-tRNA, the mutant tRNA is able to transfer valine into a polypeptide chain, albeit at a reduced level. The A76G mutation does not affect binding of Val-or NAcVal-tRNA CCG to the A-or P-sites as shown by efficient peptide bond formation, although the donor activity of the mutant NAcVal-tRNA CCG in the peptidyl transfer reaction is slightly reduced compared with wild-type NAcVal-tRNA. Translocation of 3-CCG-tRNA from the Pto the E-site is not significantly influenced. However, the A76G mutation drastically inhibits translocation of peptidyl-tRNA G 76 from the ribosomal A-site to the Psite, which apparently explains its failure to support cell-free protein synthesis. Our results indicate that the identity of the 3-terminal nucleotide of tRNA is critical for tRNA movement in the ribosome.
Biochemistry, 1998
Unmodified uridines have been randomly replaced by 4-thiouridines in transfer RNA Phe (tRNA Phe ) transcribed in a T7 RNA polymerase system. These 4-thiouridines serve as conjugation sites for attachment of the cleavage reagent 5-iodoacetamido-1,10-o-phenanthroline (IoP). In a reducing environment, when complexed with Cu 2+ , 1,10-o-phenanthroline causes cleavage of nearby nucleic acids. We show here that tRNA-phenanthroline (tRNA-oP) conjugates, when bound at the P-site of 70S ribosomes and 30S ribosomal subunits, caused cleavage of ribosomal RNA (rRNA) mainly in domains I and II of 16S rRNA. Some positions were cleaved only when tRNA-oP was bound to 70S ribosomes or to 30S ribosomal subunits. In domain I, most cleavage sites occurred in or near the 530 pseudoknot region. In domain II, most nucleotides cleaved were near the 690 region and the 790 region. The only positions cleaved in domain III were near the 1050 region. There were no discernible nucleotides cleaved near the 1400 (decoding) region. Our results corroborated results of others, which have shown these sites to be protected from chemical modification by tRNA binding or to be cross-linked to P-site-bound tRNA. Use of cleavage reagents tethered to tRNA provides evidence for additional regions of rRNA that may be proximal to bound tRNA.
Nucleic Acids Research, 2013
The accuracy of pairing of the anticodon of the initiator tRNA (tRNA fMet ) and the initiation codon of an mRNA, in the ribosomal P-site, is crucial for determining the translational reading frame. However, a direct role of any ribosomal element(s) in scrutinizing this pairing is unknown. The P-site elements, m 2 G966 (methylated by RsmD), m 5 C967 (methylated by RsmB) and the C-terminal tail of the protein S9 lie in the vicinity of tRNA fMet . We investigated the role of these elements in initiation from various codons, namely, AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG with tRNA fMet CAU (tRNA fMet with CAU anticodon); CAC and CAU with tRNA fMet GUG ; UAG with tRNA fMet CUA ; UAC with tRNA fMet GUA ; and AUC with tRNA fMet GAU using in vivo and computational methods. Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2-to 3.6-fold). Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, . Also, the S9 tail suppressed initiation with tRNA fMet CUA lacking the 3GC base pairs in the anticodon stem. These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.