A New Factor from Escherichia coli Affects Translocation of mRNA

Ganoza, M. Clelia; Cunningham, Carol C.; Rm, Green

doi:10.1074/jbc.270.44.26377

Cited by 13 publications

(20 citation statements)

References 31 publications

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“…Ganoza et al [7] and Green et al [26] demonstrated that W facilitated the release of tRNAs from the E site and that this increased cognate binding at the A site. Certainly RbbA and W may act together as a functional homologue to yeast EF-3.…”

Section: Discussionmentioning

confidence: 99%

“…An active mechanism for the ejection of the E-site-bound deacyl-tRNA that forms after peptide bond synthesis was first proposed to be mediated by a soluble protein that is essential to reconstitute synthesis from purified E. coli components [6,7]. Interestingly, a fungi-specific ATPase, EF-3, has more recently been reported to accelerate the rate of deacyl-tRNA ejection from Saccharomyces cerevisiae ribosomes [8,9].…”

mentioning

confidence: 99%

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Functional interactions of an Escherichia coli ribosomal ATPase

Kiel¹,

Ganoza²

2001

Eur J Biochem

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The gene encoding ribosome-bound ATPase (RbbA), which occurs bound to 70S ribosomes and 30S subunits, has been identified. The amino-acid sequence of RbbA reveals the presence of two ATP-binding domains in the N-terminal half of the protein. RbbA harbors an intrinsic ATPase activity that is stimulated by both 70S ribosomes and 30S subunits. Here we show that purified recombinant RbbA markedly stimulates polyphenylalanine synthesis in the presence of the elongation factors Tu and G (EF-Tu and EF-G) and that the hydrolysis of ATP by RbbA is required to stimulate synthesis. RbbA is reported to have affinity for EF-Tu but not for EF-G. Additionally, RbbA copurifies with 30S ribosomal subunits and can be crosslinked to the ribosomal protein S1. Studies using a spectrum of antibiotics, including some of similar function, revealed that hygromycin, which binds to the 30S subunit, has a significant effect on the ATPase activity and on the affinity of RbbA for ribosomes. A possible role for RbbA in protein-chain elongation is proposed.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Functional interactions of an Escherichia coli ribosomal ATPase

Kiel¹,

Ganoza²

2001

Eur J Biochem

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“…The action of EFG may be preceded by movement of the deacyltRNA from the P site to the E site (163). This reaction is stimulated strongly by the RbbA protein, but it is not certain whether the RbbA protein ejects tRNAs only from the E site after peptide bond synthesis or from the P site also (57). In the former case, EFG may mediate translocation both of the deacyl-tRNA from the P site to the E site and of the peptidyltRNA from the A site to the P site.…”

Section: Translocationmentioning

confidence: 99%

“…Fungi have another elongation factor, eEF3, which is a ribosomedependent ATPase (29). This reaction is analogous to that first discovered in E. coli and called W, which ejects tRNAs from 70S ribosomes (57,58,74). (The name of the W protein [a truncated version of RbbA] was changed to reflect the fact that the protein is a ribosome-bound ATPase.)…”

Section: Decodingmentioning

confidence: 99%

Evolutionary Conservation of Reactions in Translation

Ganoza

Kiel

Aoki

2002

Microbiol Mol Biol Rev

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SUMMARY Current X-ray diffraction and cryoelectron microscopic data of ribosomes of eubacteria have shed considerable light on the molecular mechanisms of translation. Structural studies of the protein factors that activate ribosomes also point to many common features in the primary sequence and tertiary structure of these proteins. The reconstitution of the complex apparatus of translation has also revealed new information important to the mechanisms. Surprisingly, the latter approach has uncovered a number of proteins whose sequence and/or structure and function are conserved in all cells, indicating that the mechanisms are indeed conserved. The possible mechanisms of a new initiation factor and two elongation factors are discussed in this context.

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“…The H. influenzae K46A YbaK mutant was prepared using Ni 2ϩ -nitrilotriacetic acid resin (Qiagen) as described before (28). E. coli elongation factor-Tu (EF-Tu) was isolated in the EF-Tu⅐GDP form from E. coli strain DH10B-T1 (Invitrogen) carrying pPROEX-HTb_EF-Tu (35,36). The protein was purified using Ni 2ϩ -nitrilotriacetic acid resin followed by removal of the His tag using the AcTEV protease (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Cys-tRNAPro Editing by Haemophilus influenzae YbaK via a Novel Synthetase·YbaK·tRNA Ternary Complex

Musier‐Forsyth

2005

Journal of Biological Chemistry

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Aminoacyl-tRNA synthetases are multidomain enzymes that often possess two activities to ensure translational accuracy. A synthetic active site catalyzes tRNA aminoacylation, while an editing active site hydrolyzes mischarged tRNAs. Prolyl-tRNA synthetases (ProRS) have been shown to misacylate Cys onto tRNA Pro , but lack a Cys-specific editing function. The synthetase-like Haemophilus influenzae YbaK protein was recently shown to hydrolyze misacylated Cys-tRNA Pro in trans. However, the mechanism of specific substrate selection by this single domain hydrolase is unknown. Here, we demonstrate that YbaK alone appears to lack specific tRNA recognition capabilities. Moreover, YbaK cannot compete for aminoacyl-tRNAs in the presence of elongation factor Tu, suggesting that YbaK acts before release of the aminoacyl-tRNA from the synthetase. In support of this idea, cross-linking studies reveal the formation of binary (ProRS⅐YbaK) and ternary (ProRS⅐YbaK⅐tRNA) complexes. The binding constants for the interaction between ProRS and YbaK are 550 nM and 45 nM in the absence and presence of tRNA Pro , respectively. These results suggest that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS⅐YbaK⅐tRNA complex.Aminoacyl-tRNA synthetases maintain the high fidelity of protein synthesis by activating specific amino acids and transferring them to cognate tRNAs in a process known as aminoacylation. However, some synthetases are known to misactivate noncognate amino acids, transferring them onto their cognate tRNAs. Therefore, to ensure accurate translation of the genetic code, these enzymes, which include members of both classes of synthetases, have evolved proofreading or editing functions (1-17). Pretransfer editing involves hydrolysis of the misactivated aminoacyl-adenylate, whereas post-transfer editing refers to specific hydrolysis of misacylated tRNA.To clear noncognate amino acids, a double-sieve mechanism was proposed, wherein larger amino acids are rejected by the aminoacylation active site (i.e. coarse sieve) while smaller amino acids are cleared in a second editing active site (i.e. fine sieve) (18,19). This hypothesis was confirmed by more recent biochemical and x-ray crystallographic studies (20). Although the exact site of pretransfer editing is still an open question in most systems (21), class I editing enzymes, including isoleucyl-tRNA synthetase, valyl-tRNA synthetase, and leucyl-tRNA synthetase, use the highly conserved connective polypeptide 1 domain to edit misacylated tRNAs (1, 2, 4 -8, 12). In contrast, the post-transfer editing domains used by class II synthetases are much more diverse. The internal editing domain of alanyl-tRNA synthetase (AlaRS) 2 and the N-terminal editing domain of threonyl-tRNA synthetase (with the exception of the archaebacterial enzymes) share sequence similarity (9, 22), while the editing domains of bacterial prolyl-tRNA synthetase (ProRS) (13) and phenylalanyl-tRNA synthetase (17) appear to be unique, sharing no sequence homology to any of the ot...

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A New Factor from Escherichia coli Affects Translocation of mRNA

Cited by 13 publications

References 31 publications

Functional interactions of an Escherichia coli ribosomal ATPase

Functional interactions of an Escherichia coli ribosomal ATPase

Evolutionary Conservation of Reactions in Translation

Cys-tRNAPro Editing by Haemophilus influenzae YbaK via a Novel Synthetase·YbaK·tRNA Ternary Complex

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