Protein factors associated with the SsrA⋅SmpB tagging and ribosome rescue complex

Karzai, A. Wali; Sauer, Robert T.

doi:10.1073/pnas.051628298

Cited by 93 publications

(98 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In Vitro Transcription of RNAs and Enzyme Assays-In vitro transcription using T7 RNA polymerase and purification of the [␣- 32 P]UTPlabeled pZBP transcript (27) on denaturing gels was performed as described (28,29). When RNA I.26 was used, the plasmid pT7-R25 (a kind gift from Dr. J. G. Belasco) was used as template (30).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Exoribonuclease R Interacts with Endoribonuclease E and an RNA Helicase in the Psychrotrophic Bacterium Pseudomonas syringae Lz4W

Rajyaguru

Klein

Sulthana

et al. 2005

Journal of Biological Chemistry

123

112

View full text Add to dashboard Cite

Endoribonuclease E, a key enzyme involved in RNA decay and processing in bacteria, organizes a protein complex called degradosome. In Escherichia coli, Rhodobacter capsulatus, and Streptomyces coelicolor, RNase E interacts with the phosphate-dependent exoribonuclease polynucleotide phosphorylase, DEAD-box helicase(s), and additional factors in an RNA-degrading complex. To characterize the degradosome of the psychrotrophic bacterium Pseudomonas syringae Lz4W, RNase E was enriched by cation exchange chromatography and fractionation in a glycerol density gradient. Most surprisingly, the hydrolytic exoribonuclease RNase R was found to co-purify with RNase E. Co-immunoprecipitation and Ni 2؉ -affinity pull-down experiments confirmed the specific interaction between RNase R and RNase E. Additionally, the DEAD-box helicase RhlE was identified as part of this protein complex. Fractions comprising the three proteins showed RNase E and RNase R activity and efficiently degraded a synthetic stem-loop containing RNA in the presence of ATP. The unexpected association of RNase R with RNase E and RhlE in an RNA-degrading complex indicates that the cold-adapted P. syringae has a degradosome of novel structure. The identification of RNase R instead of polynucleotide phosphorylase in this complex underlines the importance of the interaction between endo-and exoribonucleases for the bacterial RNA metabolism. The physical association of RNase E with an exoribonuclease and an RNA helicase apparently is a common theme in the composition of bacterial RNAdegrading complexes.

show abstract

Section: Methodsmentioning

confidence: 99%

“…Involvement of RNase R with other proteins and/or RNA in a putative ribosome rescue complex has also been discussed (32), but its interaction with the degradosome organizer RNase E was not known until now. Instead, RNase E was shown to co-purify with PNPase (1-5, 12, 13).…”

Section: P Syringae Rnase R Is In a Complex With Rnase E Andmentioning

confidence: 99%

Exoribonuclease R Interacts with Endoribonuclease E and an RNA Helicase in the Psychrotrophic Bacterium Pseudomonas syringae Lz4W

Rajyaguru

Klein

Sulthana

et al. 2005

Journal of Biological Chemistry

123

112

View full text Add to dashboard Cite

show abstract

“…SmpB, a dedicated tmRNA binding protein (6), mediates interactions with the ribosome, some of which are normally made by tRNA itself (7), whereas alanine tRNA synthetase and elongation factor Tu mediate aminoacylation and ribosome delivery, respectively (7,8). Ribosomal protein S1 copurifies with tmRNA-SmpB complexes, crosslinks to tmRNA both in the presence and absence of ribosomes in vitro, and binds to tmRNA in vitro (9)(10)(11), but its role in tmRNA function is unclear.…”

mentioning

confidence: 99%

Ribosomal protein S1 binds mRNA and tmRNA similarly but plays distinct roles in translation of these molecules

McGinness

Sauer²

2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Ribosomes stalled during protein synthesis can be rescued by tmRNA, which acts first as a tRNA and then as an mRNA to direct addition of a C-terminal degradation tag to the nascent polypeptide. Ribosomal protein S1 binds tmRNA, but its functional role in tmRNA-mediated tagging is uncertain. To probe interactions between S1 and tmRNA, truncated variants missing one or more of the six contiguous S1 domains were studied. The third S1 domain (R1) plays a critical role in binding tmRNA and mRNA but requires additional N-or C-terminal S1 domains. The binding of S1 and its fragments to tmRNA and mRNA is positively cooperative, and the essential role of the R1 domain may be to mediate protein-protein interactions. Overproduction of N-terminal fragments of S1 in Escherichia coli displaces endogenous S1 from ribosomes, inhibits general protein synthesis, and slows growth but causes little if any disruption of tmRNA-mediated tagging. Moreover, tagging of proteins translated from model mRNAs with either no or an increased requirement for S1 is indistinguishable. These results raise the possibility that S1 plays little or no role in tmRNAmediated tagging.ssrA tagging ͉ trans-translation ͉ ribosome rescue ͉ protein-RNA interactions

show abstract

“…RNase R Interacts with SmpB and tmRNA-It has been reported that a small amount of RNase R is present in a large complex with tmRNA and its associated protein, SmpB (18). Inasmuch as SmpB has a molecular mass close to 20 kDa (18,22), it was a possible candidate for the protein that was crosslinked to RNase R. To test this possibility, His 6 and 2ϫFLAG sequences were fused to the N termini of chromosomal RNase R and SmpB, respectively, by recombineering (16).…”

Section: Resultsmentioning

confidence: 99%

“…Associated proteins and RNAs were analyzed by Western blotting and Northern blotting, respectively. Overexpression and Purification of SmpB and tmRNA-The tandem smpB and ssrA genes (18) were amplified by PCR with primers S1 and S2 (supplemental Table S1). The PCR product was purified and digested with NdeI and BamHI and cloned into pET15b.…”

Section: Pulldown Of Rnase R From Extracts-mg1655(seq)rphmentioning

confidence: 99%

A Novel Mechanism for Ribonuclease Regulation

Liang

Deutscher

2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

The amount of RNase R, an important degradative exoribonuclease, increases 3-10-fold under a variety of stress conditions. This elevation is due to posttranslational regulation in which the highly unstable RNase R protein becomes stabilized during stress. Here we identify two components of the transtranslation machinery, transfer-messenger RNA (tmRNA) and SmpB, that are responsible for the short half-life of RNase R in exponential phase cells. The absence of either lengthens the half-life of RNase R in vivo >6-fold. SmpB directly interacts with RNase R in vitro and is stimulated by tmRNA. The C-terminal region of RNase R, encompassing its basic region and adjacent S1 domain are required for the interaction; their removal eliminates binding and stabilizes RNase R in vivo. However, the binding of SmpB and tmRNA does not alter RNase R activity. These data define a previously unknown regulatory process in which the stability of an RNase is determined by its interaction with an RNA and an RNA-associated protein.RNase R is a processive 3Ј-to 5Ј-exoribonuclease (1) that in Escherichia coli plays an important role in multiple aspects of RNA metabolism including turnover of mRNA (2, 3) and quality control of stable RNAs (4, 5). The catalytic properties of RNase R are unusual because it is able to digest structured RNAs in the absence of an added RNA helicase (1, 6, 7). As a consequence, RNase R, together with polynucleotide phosphorylase and its associated RNA helicase, are the primary RNases responsible for degradation of structured RNAs. In fact, cells lacking both RNase R and polynucleotide phosphorylase are inviable, and fragments of rRNA and structured mRNAs accumulate in their absence (2,5,8).The amount of RNase R in E. coli increases 3-10-fold during cold shock (9, 10), stationary phase (10, 11), and other stress conditions (10). The molecular mechanisms that underlie these changes in RNase R are of considerable interest inasmuch as little is known about regulation of RNase levels in cells and how this may impact RNA metabolism. In recent studies from our laboratory, it was shown that the elevation of RNase R is largely due to posttranslational regulation (12). RNase R was found to be a highly unstable protein in exponential phase with a halflife of ϳ10 min, whereas it is stabilized under stress conditions, leading to its relative elevation (12). However, the factors or processes responsible for the instability of RNase R are not understood.Here, we show that two components of trans-translation, tmRNA 2 and its associated protein, SmpB, are required for the instability of RNase R. In the absence of either, RNase R levels in exponential phase cells increase markedly, and the half-life of RNase R increases from ϳ10 min to more than 60 min. We also show that SmpB and tmRNA interact with RNase R both in vitro and in vivo and that the C-terminal region of RNase R is required for this interaction. The half-life of truncated RNase R lacking its C-terminal basic region and S1 domain increases dramatically when compared wi...

show abstract

Protein factors associated with the SsrA⋅SmpB tagging and ribosome rescue complex

Cited by 93 publications

References 23 publications

Exoribonuclease R Interacts with Endoribonuclease E and an RNA Helicase in the Psychrotrophic Bacterium Pseudomonas syringae Lz4W

Exoribonuclease R Interacts with Endoribonuclease E and an RNA Helicase in the Psychrotrophic Bacterium Pseudomonas syringae Lz4W

Ribosomal protein S1 binds mRNA and tmRNA similarly but plays distinct roles in translation of these molecules

A Novel Mechanism for Ribonuclease Regulation

Contact Info

Product

Resources

About