Mutations at Positions 13 and/or 914 in Escherichia coli 16 S Ribosomal RNA Interfere with the Initiation of Protein Synthesis

Pinard, Robert; Payant, Catherine; Brakier‐Gingras, Léa

doi:10.1021/bi00029a038

Cited by 13 publications

(5 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1). It was previously shown that the U13A mutation causes a small increase in the binding of a probe complementary to the 915-921 region and in the reactivity of G917 to kethoxal (Pinard et al 1995). The increased accessibility of residues within the central pseudoknot indicates that the U13A mutation destabilizes helix 2, making this pseudoknot more flexible.…”

Section: Discussionmentioning

confidence: 99%

A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome

Bart

Théberge-Julien

Cunningham³

et al. 2005

RNA

View full text Add to dashboard Cite

The conserved 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) interacts with helix 24 of 16S rRNA and also with helix 67 of 23S rRNA, forming the intersubunit bridge B2c, proximal to the decoding center. In previous studies, we investigated how the interaction between the 900 tetraloop and helix 24 participates in subunit association and translational fidelity. In the present study, we investigated whether the 900 tetraloop is involved in other undetected interactions with different regions of the Escherichia coli 16S rRNA. Using a genetic complementation approach, we selected mutations in 16S rRNA that compensate for a 900 tetraloop mutation, A900G, which severely impairs subunit association and translational fidelity. Mutations were randomly introduced in 16S rRNA, using either a mutagenic XL1-Red E. coli strain or an error-prone PCR strategy. Gain-offunction mutations were selected in vivo with a specialized ribosome system. Two mutations, the deletion of U12 and the U12C substitution, were thus independently selected in helix 1 of 16S rRNA. This helix is located in the vicinity of helix 27, but does not directly contact the 900 tetraloop in the crystal structures of the ribosome. Both mutations correct the subunit association and translational fidelity defects caused by the A900G mutation, revealing an unanticipated functional interaction between these two regions of 16S rRNA.

show abstract

Section: Discussionmentioning

confidence: 99%

A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome

Bart

Théberge-Julien

Cunningham³

et al. 2005

RNA

View full text Add to dashboard Cite

show abstract

“…Plasmid pUC188G32 (42) and pLRCAT (43) were used for the in vitro transcription of the T4 gene 32 and the gene coding for chloramphenicol acetyl transferase (CAT), 1 respectively, and the mRNAs produced were used for toeprinting and filter-binding studies.…”

Section: Construction Of Plasmids Used In Thismentioning

confidence: 99%

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

Robert¹,

Brakier‐Gingras

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

“…The increased reactivity in the synthetic ribosomes (Figure 2) suggests that the structure of the central pseudoknot is less stable than that in natural RNA. The central pseudoknot has been shown to be necessary for efficient translation (Brink et al, 1993;Pinard et al, 1995). Although essential functions are maintained in 30S subunits reconstituted with in Vitrotranscribed RNA (Denman et al, 1989b;Cunningham et al, 1991), these ribosomes have a reduced activity as tested in in Vitro translation assays compared to ribosomes isolated from cells.…”

Section: Discussionmentioning

confidence: 99%

Conformational Analysis of Escherichia coli 30S Ribosomes Containing the Single-Base Mutations G530U, U1498G, G1401C, and C1501G and the Double-Base Mutation G1401C/C1501G

et al. 1997

View full text Add to dashboard Cite

Biochemical and genetic studies have pointed out the importance of several sites in 16S ribosomal RNA of Escherichia coli in the decoding process. These sites consist of the core of the decoding center (1400/1500 region) and two other segments (530 and 1050/1200 regions). To detect a possible structural link between these functionally related regions, we analyzed their sensitivity to conformational changes induced by mutations which are located in each of these regions and are known to affect the decoding process. The conformations of five segments of 16S rRNA (1-106, 406-569, 780-978, 997-1247, and 1334-1519) were analyzed by chemical probing of 30S ribosomes containing the following mutations: G530U, U1498G, G1401C, C1501G, and G1401C/C1501G. Ribosomes reconstituted with natural wild-type 16S RNA showed only minor conformational differences with respect to ribosomes isolated from cells. When 16S RNA made in vitro replaced natural 16S RNA, a slightly looser conformation of the central core region was found. Mutant ribosomes made by reconstitution with mutant 16S RNA made in vitro showed conformational effects which were in all cases localized to the region of secondary structure surrounding the site of mutation. Although the core of the decoding center (1400/1500 region) and the two other sites (530 and 1050/1200 regions) participating in the decoding function have been functionally linked, our data indicate that they are structurally independent. They also provide evidence for an unusual structure of the 1400/1500 decoding center, possibly involving noncanonical interactions. Furthermore, the absence of any conformational effect induced by the G530U mutation except at the site of mutation itself points to its direct, as opposed to indirect, involvement in the decoding function of the ribosome.

show abstract

Mutations at Positions 13 and/or 914 in Escherichia coli 16 S Ribosomal RNA Interfere with the Initiation of Protein Synthesis

Cited by 13 publications

References 27 publications

A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome

A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome

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

Conformational Analysis of Escherichia coli 30S Ribosomes Containing the Single-Base Mutations G530U, U1498G, G1401C, and C1501G and the Double-Base Mutation G1401C/C1501G

Contact Info

Product

Resources

About