Ribosome Initiation Complex Formation with the Pseudoknotted α Operon Messenger RNA

Spedding, Gary; Gluick, Thomas C.; Draper, David E.

doi:10.1006/jmbi.1993.1067

Cited by 44 publications

(47 citation statements)

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“…The extended toeprints were sensitive to reverse transcriptase concentration as well as reaction temperature (Ringquist et al 1993a), suggesting that they represent weak interactions that are sensitive to reverse transcriptase activity. In contrast, the + 16 toeprints usually observed for ribosome binding were unaffected by these variables (Spedding et al 1993;Ringquist et al 1993b). Moreover, three-dimensional models of the ribosome have indicated the presence of a large channel between the 30S and 50S subunits (Frank et al 1992).…”

Section: Discussionmentioning

confidence: 89%

“…Baron et al (1993b) observed enhanced binding of SELB to the SECIS in the presence of GTP and Sec-tRNA^^". These results differ possibly because the mobility-shift assay employed by Baron et al (1993b) and the toeprint assay may examine different kinetic properties of the SELB-mRNA complex (Ringquist et al 1993b;Spedding et al 1993); perhaps, the mobility-shift assay is more sensitive than toeprinting to changes in the off-rate of the SELB-mRNA or SELB-tRNA-mRNA complex.…”

Section: Toepimting Of Selb At the Secismentioning

confidence: 98%

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Recognition of the mRNA selenocysteine insertion sequence by the specialized translational elongation factor SELB.

Ringquist¹,

Schneider²,

Gibson³

et al. 1994

Genes Dev.

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In Escherichia coli the unusual amino acid selenocysteine is incorporated cotranslationally at an in-frame UGA codon. Incorporation of selenocysteine relies, in part, on the interaction between a specialized elongation factor, the SELB protein, and a cis-acting element within the mRNA. Boundary and toeprint experiments illustrate that the SELB-GTP-Sec-tRNA^''' ternary complex binds to the selenoprotein encoding mRNAs fdhF and fdnG, serving to increase the concentration of SELB and Sec-tRNA**'' on these mRNAs in vivo. Moreover, toeprint experiments indicate that SELB recognizes the ribosome-bound message and that, upon binding, SELB may protrude out of the ribosomal-mRNA track so as to approach the large ribosomal subunit. The results place the mRNA-bound SELB-GTP-Sec-tRNA^^'' ternary complex at the selenocysteine codon (as expected) and suggest a mechanism to explain the specificity of selenocysteine insertion. Cis-acting mRNA regulatory elements can tether protein factors to the translation complex during protein synthesis.

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Section: Discussionmentioning

confidence: 89%

Section: Toepimting Of Selb At the Secismentioning

confidence: 98%

Recognition of the mRNA selenocysteine insertion sequence by the specialized translational elongation factor SELB.

Ringquist¹,

Schneider²,

Gibson³

et al. 1994

Genes Dev.

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“…A toeprint assay allows the detection of canonical initiation complexes, because an mRNA-bound 30 S, in presence of the initiator tRNA (tRNA fMet ), blocks the extension of a primer by the reverse transcriptase. This stop, called the toeprint signal, occurs at position ϩ16 and also, at a lower extent, at ϩ17 on the mRNA (the A of the initiator codon is ϩ1) (45,52,53). Examples of typical toeprint experiments are presented in Fig.…”

Section: Resultsmentioning

confidence: 99%

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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“…The resuspended ribosomes were centrifuged at 52,000 rpm (Beckman Ti55.2 rotor) for 140 min, and the pellet material was resuspended overnight at 0°C in buffer A. Ribosomes were salt washed once by centrifugation through an equal volume of buffer A plus 15% (w/v) sucrose and 500 mM NH 4 Cl; resuspended ribosomes (buffer A or buffer A with 1.1 mM Mg(OAc) 2 ) were stored frozen at Ϫ70°C. 30 S subunits were prepared by sucrose gradient sedimentation of ribosomes in buffer A with 1.1 mM Mg(OAc) 2 , as described (10).…”

Section: Methodsmentioning

confidence: 99%

“…1B). Kinetic studies of initiation complex formation suggested that the mRNA adopted two conformations, both capable of binding 30 S subunits, but only the "active" conformation supported formation of the ternary initiation complex (10). In further studies of S4 binding kinetics, it appeared that the repressor bound only the "inactive" conformation and trapped 30 S subunits in a dead-end complex unable to bind tRNA f Met (5).…”

Section: Synthesis Of Nearly All Ribosomal Proteins In Escherichia Colimentioning

confidence: 99%

Translational Repression of the Escherichia coli α Operon mRNA

Schlax¹,

Xavier²,

Gluick³

et al. 2001

Journal of Biological Chemistry

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Ribosomal protein S4 represses synthesis of the four ribosomal proteins (including itself) in the Escherichia coli ␣ operon by binding to a nested pseudoknot structure that spans the ribosome binding site. A model for the repression mechanism previously proposed two unusual features: (i) the mRNA switches between conformations that are "active" or "inactive" in translation, with S4 as an allosteric effector of the inactive form, and (ii) S4 holds the 30 S subunit in an unproductive complex on the mRNA ("entrapment"), in contrast to direct competition between repressor and ribosome binding ("displacement"). These two key points have been experimentally tested. First, it is found that the mRNA pseudoknot exists in an equilibrium between two conformers with different electrophoretic mobilities. S4 selectively binds to one form of the RNA, as predicted for an allosteric effector; binding of ribosomal 30 S subunits is nearly equal in the two forms. Second, we have used S4 labeled at a unique cysteine with either of two fluorophores to characterize its interactions with mRNA and 30 S subunits. Equilibrium experiments detect the formation of a specific ternary complex of S4, mRNA pseudoknot, and 30 S subunits. The existence of this ternary complex is unambiguous evidence for translational repression of the ␣ operon by an entrapment mechanism.

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Ribosome Initiation Complex Formation with the Pseudoknotted α Operon Messenger RNA

Cited by 44 publications

References 0 publications

Recognition of the mRNA selenocysteine insertion sequence by the specialized translational elongation factor SELB.

Recognition of the mRNA selenocysteine insertion sequence by the specialized translational elongation factor SELB.

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

Translational Repression of the Escherichia coli α Operon mRNA

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