Allosteric mechanism for translational repression in the Escherichia coli alpha operon.

Spedding, Gary; Draper, David E.

doi:10.1073/pnas.90.10.4399

Cited by 61 publications

(50 citation statements)

References 23 publications

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“…There are other examples in which RNA structures containing SD were reported to be unfolded at higher temperature resulting in faster toeprint complex formation (Spedding and Draper 1993;Brunel et al 1995). In the present system, melting of mRNA structure by heat was directly linked with the biological function of its product protein 32 .…”

Section: Discussionmentioning

confidence: 99%

Translational induction of heat shock transcription factor sigma 32: evidence for a built-in RNA thermosensor

Morita¹,

Tanaka²,

Kodama³

et al. 1999

Genes & Development

272

260

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Induction of heat shock proteins in Escherichia coli is primarily caused by increased cellular levels of the heat shock -factor 32 encoded by the rpoH gene. Increased 32 levels result from both enhanced synthesis and stabilization. Previous work indicated that 32 synthesis is induced at the translational level and is mediated by the mRNA secondary structure formed within the 5-coding sequence of rpoH, including the translation initiation region. To understand the mechanism of heat induction of 32 synthesis further, we analyzed expression of rpoH-lacZ gene fusions with altered stability of mRNA structure before and after heat shock. A clear correlation was found between the stability and expression or the extent of heat induction. Temperature-melting profiles of mRNAs with or without mutations correlated well with the expression patterns of fusion genes carrying the corresponding mutations in vivo. Furthermore, temperature dependence of mRNA-30S ribosome-tRNA f Met complex formation with wild-type or mutant mRNAs in vitro agreed well with that of the expression of gene fusions in vivo. Our results support a novel mechanism in which partial melting of mRNA secondary structure at high temperature enhances ribosome entry and translational initiation without involvement of other cellular components, that is, intrinsic mRNA stability controls synthesis of a transcriptional regulator.

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Section: Discussionmentioning

confidence: 99%

Translational induction of heat shock transcription factor sigma 32: evidence for a built-in RNA thermosensor

Morita¹,

Tanaka²,

Kodama³

et al. 1999

Genes & Development

272

260

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“…It directly recognizes the base of a multiheaded helix of the SSU rRNA to facilitate correct RNA folding and assembly with the other SSU proteins (27,33). It also directly associates with a pseudoknot structure in the regulatory sequences of the mRNA of its own operon, thereby modulating expression of itself and other ribosomal proteins (32). The S4 ribosomal protein, therefore, is a protein that binds directly to two distinct RNAs of diverse sequence and structure with remarkable versatility.…”

Section: Discussionmentioning

confidence: 99%

Imp3p and Imp4p, Two Specific Components of the U3 Small Nucleolar Ribonucleoprotein That Are Essential for Pre-18S rRNA Processing

Lee¹,

Baserga

1999

Molecular and Cellular Biology

128

158

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The function of the U3 small nucleolar ribonucleoprotein (snoRNP) is central to the events surrounding prerRNA processing, as evidenced by the severe defects in cleavage of pre-18S rRNA precursors observed upon depletion of the U3 RNA and its unique protein components. Although the precise function of each component remains unclear, since U3 snoRNA levels remain unchanged upon genetic depletion of these proteins, it is likely that the proteins themselves have significant roles in the cleavage reactions. Here we report the identification of two previously undescribed protein components of the U3 snoRNP, representing the first snoRNP components identified by using the two-hybrid methodology. By screening for proteins that physically associate with the U3 snoRNP-specific protein, Mpp10p, we have identified Imp3p (22 kDa) and Imp4p (34 kDa) (named for interacting with Mpp10p). The genes encoding both proteins are essential in yeast. Genetic depletion reveals that both proteins are critical for U3 snoRNP function in pre-18S rRNA processing at the A0, A1, and A2 sites in the pre-rRNA. Both Imp proteins associate with Mpp10p in vivo, and both are complexed only with the U3 snoRNA. Conservation of RNA binding domains between Imp3p and the S4 family of ribosomal proteins suggests that it might associate with RNA directly. However, as with other U3 snoRNP-specific proteins, neither Imp3p nor Imp4p is required for maintenance of U3 snoRNA integrity. Imp3p and Imp4p are therefore novel protein components specific to the U3 snoRNP with critical roles in pre-rRNA cleavage events.Eukaryotic ribosomes are large ribonucleoproteins (RNPs) composed of four rRNAs and dozens of ribosomal proteins. Three of the four RNA components are processed from a single polycistronic transcript, undergo extensive nucleotide modification, and assemble with the appropriate proteins in the cell nucleolus. In the yeast Saccharomyces cerevisiae, the small-subunit (SSU) rRNA (18S) and the large-subunit (LSU) rRNAs (5.8S and 25S) are processed from the nascent 35S transcript via a series of cleavage steps (Fig. 1A). Successive cleavages at the 5Ј end of the precursor rRNA yield the 33S and 32S transcripts, while subsequent cleavage at the A2 site separates the precursors of the SSU rRNA from those of the LSU rRNA. The 20S pre-rRNA is matured to 18S rRNA, while the LSU precursors are terminally processed to the 5.8S and 25S rRNAs.Several small nucleolar RNPs (snoRNPs) are required for the cleavage steps that generate the mature 18S rRNA (reviewed in reference 35). Of these, the most widely studied is the U3 snoRNP. In yeast, genetic depletion of the U3 snoRNA results in a deficiency in processing at the A0, A1, and A2 sites, causing a sharp decrease in 20S and 18S rRNA levels and an apparent increase in the presence of the 23S precursor and the 35S nascent transcript (Fig. 1B). A role for the U3 snoRNP in pre-rRNA cleavage events has also been observed in metazoans. Oligonucleotide-mediated depletion of the U3 snoRNA in vivo in Xenopus laevis oocytes an...

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“…Experiments first suggesting that the ␣ mRNA exists in two forms were based on a toeprint assay (5,10), in which a 30 S subunit⅐ tRNA f Met ⅐mRNA initiation complex stops reverse transcription at G110 (Fig. 1A), and a 30 S subunit⅐S4⅐mRNA complex induces transcriptase termination at the downstream edge of the pseudoknot, U120-G125.…”

Section: Discussionmentioning

confidence: 99%

“…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). This repression strategy has two novel aspects.…”

Section: Synthesis Of Nearly All Ribosomal Proteins In Escherichia Colimentioning

confidence: 97%

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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Allosteric mechanism for translational repression in the Escherichia coli alpha operon.

Cited by 61 publications

References 23 publications

Translational induction of heat shock transcription factor sigma 32: evidence for a built-in RNA thermosensor

Translational induction of heat shock transcription factor sigma 32: evidence for a built-in RNA thermosensor

Imp3p and Imp4p, Two Specific Components of the U3 Small Nucleolar Ribonucleoprotein That Are Essential for Pre-18S rRNA Processing

Translational Repression of the Escherichia coli α Operon mRNA

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