Translational feedback regulation of the gene for L35 in Escherichia coli requires binding of ribosomal protein L20 to two sites in its leader mRNA: A possible case of ribosomal RNA???messenger RNA molecular mimicry

Guillier, Maude; Allemand, Frédéric; Raibaud, Sophie; Dardel, Frédéric; Springer, Mathias; Chiaruttini, Claude

doi:10.1017/s1355838202029084

Cited by 42 publications

(73 citation statements)

References 18 publications

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“…in the absence of divalent cations (Mg 2ϩ ), which can sometime mediate unspecific aggregation of RNA at high concentrations. This is of great potential relevance in the context of translational control of the IF3 operon for which two target sites for L20 have been identified within the operator region upstream of the rpmI translation start, which are both similar to the rRNAbinding site of L20C (22). Interestingly, these two sites are able to bind L20 independently, i.e.…”

Section: Discussionmentioning

confidence: 94%

“…mutations that affect L20 binding at one site do not prevent binding to the other site. However, simultaneous binding at both sites appear to be required for the negative feedback control of rpmI (22). This led to the hypothesis that two L20 molecules could be required for the translational repression on the rpmI operator.…”

Section: Discussionmentioning

confidence: 99%

“…The radius of gyration and the intensity at the origin were derived from a Guinier analysis of the scattering intensities at s Ͻ s max ϭ x/(2R g ) (32) (24), numbering is that of the E. coli 23 S rRNA. oRNA, secondary structure of the L20-binding site 2 on the rpmI translational operator (22). Outlined residues were added to increase the pairing stability.…”

Section: Methodsmentioning

confidence: 99%

“…(i) The C-terminal domain suffices to repress rpmI expression (23). (ii) In the crystal structure of D. radiodurans 50 S subunit (24), the C-terminal domain of L20 interacts tightly with a discrete region of the 23 S rRNA, at the junction between helices 40 and 41 (25), which exhibits similarities with the rpmI translational operator (22). (iii) It was suspected that the strongly basic and unfolded N-terminal domain would make spurious electrostatic contacts with the RNA fragments, yielding to nonspecific aggregation of the sample, a phenomenon indeed observed in preliminary experiments (not shown).…”

Section: Structural Analysis Of L20c the Translational Control Domainmentioning

confidence: 99%

“…They are composed of two independent structures, a pseudoknot structure that involves long range RNA-RNA interactions and overlaps the rpmI ribosome-binding site and initiation codon (site 1) (21), and an imperfect stem structure located in the infC-rpmI intergenic region (site 2) (22). Interestingly, both sites are apparently independently bound by L20, and both are required for repression of expression of L35 and L20 (22). This peculiar "dual-site" organization of the operator raises a number of questions.…”

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confidence: 99%

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How Bacterial Ribosomal Protein L20 Assembles with 23 S Ribosomal RNA and Its Own Messenger RNA

Raibaud

Vachette

Guillier

et al. 2003

Journal of Biological Chemistry

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In bacteria, the expression of ribosomal proteins is often feedback-regulated at the translational level by the binding of the protein to its own mRNA. This is the case for L20, which binds to two distinct sites of its mRNA that both resemble its binding site on 23 S rRNA. In the present work, we report an NMR analysis of the interaction between the C-terminal domain of L20 (L20C) and both its rRNA-and mRNA-binding sites. Changes in the NMR chemical shifts of the L20C backbone nuclei were used to show that the same set of residues are modified upon addition of either the rRNA or the mRNA fragments, suggesting a mimicry at the atomic level. In addition, small angle x-ray scattering experiments, performed with the rRNA fragment, demonstrated the formation of a complex made of two RNAs and two L20C molecules. A low resolution model of this complex was then calculated using (i) the rRNA/L20C structure in the 50 S context and (ii) NMR and small angle x-ray scattering results. The formation of this complex is interesting in the context of gene regulation because it suggests that translational repression could be performed by a complex of two proteins, each interacting with the two distinct L20-binding sites within the operator.Ribosome assembly is a complex process in which proteins must assemble onto the ribosomal RNA in an ordered fashion. This process has been extensively analyzed for bacterial ribosomal subunits, using in vitro reconstitution studies. In the case of Escherichia coli large 50 S subunit, it has been shown that nine "core proteins" L1, L3, L4, L9, L10, L11, L20, L23, and L24 bind directly to the 23 S rRNA, independently of other proteins (1, 2). The other 50 S ribosomal proteins depend upon those primary binders for attachment to the large ribosomal subunit. Interestingly, several of these core proteins, namely L1, L4, L10, and L20, are also involved in the feedback regulation that allows the coordinated expression of the various ribosomal components (3-8). A similar situation is also observed for the small ribosomal subunit where regulatory ribosomal proteins are also primary 16 S rRNA binders.Most bacterial ribosomal protein genes are clustered in polycistronic operons, the expression of which is controlled at the translational level (9 -11). One cistron encodes a regulatory ribosomal protein that binds to the messenger RNA, thereby shutting off the translation of the downstream ribosomal protein cistrons. In the classical Nomura model, it is assumed that the protein-binding site on the mRNA, the translational operator, is structurally similar to its binding site on the rRNA. Molecular mimicry between the two target RNA sites would then be used to adjust the translation of ribosomal proteins to the level of transcription of the rRNA; in the presence of excess unbound rRNA, the repressor ribosomal protein would be displaced from its mRNA, thereby allowing translation to proceed. So far, in E. coli, the molecular mimicry between the translational operator on the mRNA and the rRNA has been demonstrate...

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Structural Analysis Of L20c the Translational Control Domainmentioning

confidence: 99%

mentioning

confidence: 99%

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How Bacterial Ribosomal Protein L20 Assembles with 23 S Ribosomal RNA and Its Own Messenger RNA

Raibaud

Vachette

Guillier

et al. 2003

Journal of Biological Chemistry

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Regulation of Ribosome Biosynthesis in Escherichia coli

Iskakova

Connell

Nierhaus

2004

Protein Synthesis and Ribosome Structure

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Mutagenesis: Site‐Specific

Walquist

Buvang

El-Gewely

2014

Encyclopedia of Life Sciences

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Site‐specific mutagenesis techniques, also known as site‐directed mutagenesis (SDM), aim to introduce precise alterations in any coding or noncoding deoxyribonucleic acid (DNA) sequence, usually in vitro . These modifications could be as small as a nucleotide or several hundreds; in one site or in multisite in the same DNA sequence. Recently, these alterations have been also developed in vivo . SDM success depends on how changes are introduced and mutant selection is done. DNA sequence analysis has to be made to verify change(s) before any biochemical or biological experiments are done. Recent methods for SDM and most used commercial kits are discussed. A list of companies offering SDM service is included. The authors have also listed software used for mutagenic oligonucleotide primer‐design. These techniques are revolutionising our understanding of the genetic and molecular mechanisms, protein structure–function relationship, protein–protein interaction, binding sites in any biological system. In addition to the academic benefits of SDM, SDM techniques have impacted biotechnology and the applied field such as engineering new enzymes, drug development, optimisation of heterologous gene expression and secretion. Key Concepts: All site‐specific alterations requiring site‐directed mutagenesis technique are done at the DNA level making it heritable modifications. Modifications done at the protein levels are not heritable. The results of these alterations are reflected in the encoded amino acids sequence of the proteins or in any targeted binding site in the DNA sequence. Several simplified Techniques are now available. Selection of the altered DNA molecules from the pool of nonmodified parental molecules is essential. DNA sequence to verify the DNA change is fundamental part of the technique. Biological and biochemical ramifications as a result of SDM are usually the purpose that SDM is done in the first place.

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Translational feedback regulation of the gene for L35 in Escherichia coli requires binding of ribosomal protein L20 to two sites in its leader mRNA: A possible case of ribosomal RNA???messenger RNA molecular mimicry

Cited by 42 publications

References 18 publications

How Bacterial Ribosomal Protein L20 Assembles with 23 S Ribosomal RNA and Its Own Messenger RNA

How Bacterial Ribosomal Protein L20 Assembles with 23 S Ribosomal RNA and Its Own Messenger RNA

Regulation of Ribosome Biosynthesis in Escherichia coli

Mutagenesis: Site‐Specific

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