Mapping the lacZ ribosome binding site by RNA footprinting

Biochemistry ◽  
1989 ◽  
Vol 28 (20) ◽  
pp. 8067-8072 ◽  
Author(s):  
George J. Murakawa ◽  
Donald P. Nierlich
Keyword(s):  
Binding Site ◽  
Ribosome Binding Site ◽  
Ribosome Binding

pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

1998 ◽  
Vol 44 (12) ◽  
pp. 1186-1192
Author(s):  
Guy Daxhelet ◽  
Philippe Gilot ◽  
Etienne Nyssen ◽  
Philippe Hoet
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Initiation Site ◽  
Chloramphenicol Acetyltransferase ◽  
Ribosome Binding Site ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Ribosome Binding

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phase of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.Key words: chloramphenicol acetyltransferase, Bacillus subtilis, postexponential gene expression, plasmid pUB110, ribosome-binding site, transcriptional promoter.

Small molecules targeting ribosome binding site of ricin toxin a subunit

Toxicon ◽  
2020 ◽  
Vol 177 ◽  
pp. S45
Author(s):  
Xiao-Ping Li ◽  
Nilgun E. Tumer ◽  
Jennifer Nielsen Kahn
Keyword(s):  
Small Molecules ◽  
Binding Site ◽  
Ribosome Binding Site ◽  
Toxin A ◽  
Ricin Toxin ◽  
Ribosome Binding ◽  
A Subunit

scholarly journals Spanning high-dimensional expression space using ribosome-binding site combinatorics

2013 ◽  
Vol 41 (9) ◽  
pp. e98-e98 ◽  
Author(s):  
Lior Zelcbuch ◽  
Niv Antonovsky ◽  
Arren Bar-Even ◽  
Ayelet Levin-Karp ◽  
Uri Barenholz ◽  
...  
Keyword(s):  
Binding Site ◽  
Ribosome Binding Site ◽  
High Dimensional ◽  
Ribosome Binding

scholarly journals Crystal structure and ligand-induced folding of the SAM/SAH riboswitch

2020 ◽  
Author(s):  
Lin Huang ◽  
Ting-Wei Liao ◽  
Jia Wang ◽  
Taekjip Ha ◽  
David M J Lilley
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Translational Regulation ◽  
Stable State ◽  
Helical Structure ◽  
Ribosome Binding Site ◽  
Stem Loop ◽  
Ribosome Binding ◽  
Induced Folding ◽  
Initiation Of Translation

Abstract While most SAM riboswitches strongly discriminate between SAM and SAH, the SAM/SAH riboswitch responds to both ligands with similar apparent affinities. We have determined crystal structures of the SAM/SAH riboswitch bound to SAH, SAM and other variant ligands at high resolution. The riboswitch forms an H-type pseudoknot structure with coaxial alignment of the stem–loop helix (P1) and the pseudoknot helix (PK). An additional three base pairs form at the non-open end of P1, and the ligand is bound at the interface between the P1 extension and the PK helix. The adenine nucleobase is stacked into the helix and forms a trans Hoogsteen–Watson–Crick base pair with a uridine, thus becoming an integral part of the helical structure. The majority of the specific interactions are formed with the adenosine. The methionine or homocysteine chain lies in the groove making a single hydrogen bond, and there is no discrimination between the sulfonium of SAM or the thioether of SAH. Single-molecule FRET analysis reveals that the riboswitch exists in two distinct conformations, and that addition of SAM or SAH shifts the population into a stable state that likely corresponds to the form observed in the crystal. A model for translational regulation is presented whereby in the absence of ligand the riboswitch is largely unfolded, lacking the PK helix so that translation can be initiated at the ribosome binding site. But the presence of ligand stabilizes the folded conformation that includes the PK helix, so occluding the ribosome binding site and thus preventing the initiation of translation.

scholarly journals Capsule depolymerase overexpression reduces Bacillus anthracis virulence

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1459-1467 ◽  
Author(s):  
Angelo Scorpio ◽  
Donald J. Chabot ◽  
William A. Day ◽  
Timothy A. Hoover ◽  
Arthur M. Friedlander
Keyword(s):  
Bacillus Anthracis ◽  
Binding Site ◽  
Consensus Sequence ◽  
Elongation Factor ◽  
Ribosome Binding Site ◽  
Start Codon ◽  
Heat Shock Protein 60 ◽  
Phagocytic Cells ◽  
Ribosome Binding ◽  
Guinea Pig Model

Capsule depolymerase (CapD) is a γ-glutamyl transpeptidase and a product of the Bacillus anthracis capsule biosynthesis operon. In this study, we examined the effect of modulating capD expression on B. anthracis capsule phenotype, interaction with phagocytic cells and virulence in guinea pigs. Transcriptional fusions of capD were made to the genes encoding heat-shock protein 60 (hsp60) and elongation factor Tu (EFTu), and to capA, a B. anthracis capsule biosynthesis gene. Translation signals were altered to improve expression of capD, including replacing the putative ribosome-binding site with a consensus sequence and the TTG start codon with ATG. CapD was not detected by immunoblotting in lysates from wild-type B. anthracis Ames but was detected in strains engineered with a consensus ribosome-binding site for capD. Strains overexpressing capD at amounts detected by immunoblotting were found to have less surface-associated capsule and released primarily lower-molecular-mass capsule into culture supernatants. Overexpression of capD increased susceptibility to neutrophil phagocytic killing and adherence to macrophages and resulted in reduced fitness in a guinea pig model of infection. These data suggest that B. anthracis may have evolved weak capD expression resulting in optimized capsule-mediated virulence.

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