Binding of initiation factor 2 and initiator tRNA to the Escherichia coli 30S ribosomal subunit induces allosteric transitions in 16S rRNA

Biochemistry ◽  
1990 ◽  
Vol 29 (35) ◽  
pp. 8144-8151 ◽  
Author(s):  
Hiroshi Wakao ◽  
Pascale Romby ◽  
Soumaya Laalami ◽  
Jean Pierre Ebel ◽  
Chantal Ehresmann ◽  
...  
FEBS Letters ◽  
1986 ◽  
Vol 202 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Claudio O. Gualerzi ◽  
Wolfgang Wintermeyer

2008 ◽  
Vol 190 (7) ◽  
pp. 2537-2545 ◽  
Author(s):  
Tracey L. Campbell ◽  
Eric D. Brown

ABSTRACT The Escherichia coli protein YjeQ is a circularly permuted GTPase that is broadly conserved in bacteria. An emerging body of evidence, including cofractionation and in vitro binding to the ribosome, altered polysome profiles after YjeQ depletion, and stimulation of GTPase activity by ribosomes, suggests that YjeQ is involved in ribosome function. The growth of strains lacking YjeQ in culture is severely compromised. Here, we probed the cellular function of YjeQ with genetic screens of ordered E. coli genomic libraries for suppressors and enhancers of the slow-growth phenotype of a ΔyjeQ strain. Screening for suppressors using an ordered library of 374 clones overexpressing essential genes and genes associated with ribosome function revealed that two GTPases, Era and initiation factor 2, ameliorated the growth and polysome defects of the ΔyjeQ strain. In addition, seven bona fide enhancers of slow growth were identified (Δtgt, ΔksgA, ΔssrA, ΔrimM, ΔrluD, ΔtrmE/mnmE, and ΔtrmU/mnmA) among 39 deletions (in genes associated with ribosome function) that we constructed in the ΔyjeQ genetic background. Taken in context, our work is most consistent with the hypothesis that YjeQ has a role in late 30S subunit biogenesis.


Author(s):  
Pascale Romby ◽  
Hiroshi Wakao ◽  
Eric Westhof ◽  
Marianne Grunberg-Manago ◽  
Bernard Ehresmann ◽  
...  

2002 ◽  
Vol 7 (6) ◽  
pp. 541-546 ◽  
Author(s):  
Sonia Delle Fratte ◽  
Chiara Piubelli ◽  
Enrico Domenici

Translational initiation factor 2 (IF2) is the largest of the 3 factors required for translation initiation in prokaryotes and has been shown to be essential in Escherichia coli. It stimulates the binding of fMet-tRNAfMet to the 30S ribosomal subunit in the presence of GTP. The selectivity is achieved through specific recognition of the tRNAfMet blocked α-amino group. IF2 is composed of 3 structural domains: N-domain, whose function is not known; G-domain, which contains the GTP/GDP binding site and the GTPase catalytic center; and C-domain, which recognizes and binds fMet-tRNAfMet. Its activity is strictly bacteria specific and highly conserved among prokaryotes. So far, antibiotics targeting IF2 function are not known, and this makes it an ideal target for new drugs with mechanisms of resistance not yet developed. A few assays have been developed in the past, which allow the detection of IF2 activity either directly or indirectly. In both instances, the assays are based on radioactive detection and do not allow for high throughput because of the need for separation or solvent extraction steps. The authors describe a novel biochemical assay for IF2 that exploits the molecular recognition of fMet-tRNAfMet by the C-domain. The assay is based on the incubation of biotinyl-IF2 with fMet-tRNAfMet and the subsequent capture of the radiolabeled complex by streptavidin-coated beads, exploiting the scintillation proximity assay (SPA) technology. The assay has been designed in an automatable, homogeneous, miniaturized fashion suitable for high-throughput screening and is rapid, sensitive, and robust to dimethyl sulfoxide (DMSO) up to 10% v/v. The assay, used to screen a limited chemical collection of about 5000 compounds and a subset of compounds originated by a 2-D substructural search, has shown to be able to detect potential IF2 inhibitors.


2010 ◽  
Vol 313 (2) ◽  
pp. 141-147
Author(s):  
Woo-Seok Song ◽  
Sang-Mi Ryou ◽  
Hong-Man Kim ◽  
Che Ok Jeon ◽  
Jong-Myung Kim ◽  
...  

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