Alteration of polynucleotide secondary structure by ribosomal protein S1.

Zwieb

2005

RNA

In bacteria, translation of mRNAs lacking stop codons produces truncated polypeptides and traps ribosomes in unproductive complexes. Potentially harmful truncated proteins are tagged with short peptides encoded by the mRNA-like domain of tmRNA and targeted for digestion by housekeeping proteases. We show that altered Escherichia coli transfer-messenger RNAs (tmRNAs) produce in vivo fusion proteins with peptide tags that extend far beyond the conventional termination signal of the wild-type tmRNA. Regions of tmRNA capable of serving as templates for protein synthesis include helix 5, as well as pseudoknots 2, 3, and 4. The removal of all six in-frame stop codons negatively affects tmRNA processing, thereby preventing translation of the 3 portion of the tRNA-like domain. These findings provide evidence that trans-translation can be accompanied by the unfolding of significant portions of the tmRNA molecule. Many of these conformational changes are likely to be required during transtranslation to maintain the ribosomal subunits in close proximity to the tmRNA for monitoring its transit.

Section: Tmrna Unfoldingmentioning

confidence: 62%

Transfer-messenger RNA unfolds as it transits the ribosome

Zwieb

2005

RNA

“…Interestingly, Hfq has been repeatedly copurified with ribosomal protein S1 as part of the bacteriophage Qb replication complex (Inouye et al 1974;Wahba et al 1974), both proteins are present in stoichiometric amounts in preparations of RNA polymerase (Sukhodolets and Garges 2003), and Hfq, S1, and RNA polymerase subunits are found as binding partners of small RNAs (N. Windbichler and R. Schroeder, unpubl.). S1 is an abundant cellular protein that is involved in mRNA binding to the small ribosomal subunit; it disrupts RNA secondary structures in vitro and unwinds mRNAs during translation initiation in vivo (Bear et al 1976;Kolb et al 1977;Subramanian 1983;Tedin et al 1997). …”

Section: Introductionmentioning

confidence: 99%

Dissecting RNA chaperone activity

Rajkowitsch¹,

Schroeder²

2007

RNA

116

145

Many RNA-binding proteins help RNAs to fold via their RNA chaperone activity. This term has been used widely without accounting for the diversity of the observed reactions, which include complex events like restructuring of misfolded catalytic RNAs, promoting the assembly of RNA-protein complexes, and mediating RNA-RNA interactions. Proteins display very diverse activities depending on the assays used to measure RNA chaperone activity. To classify proteins with this activity, we compared three exemplary proteins from E. coli, host factor Hfq, ribosomal protein S1, and the histone-like protein StpA for their abilities to promote two simple reactions, RNA annealing and strand displacement. The results of a FRET-based assay show that S1 promotes only RNA strand displacement while Hfq solely enhances RNA annealing. StpA, in contrast, is active in both reactions. To test whether the two activities can be assigned to different domains of the bipartite-structured StpA, we assayed the purified N-and C-terminal domains separately. While both domains are unable to promote RNA annealing, we can attribute the RNA strand displacement activity of StpA to the C-terminal domain. Correlating with their RNA annealing activities, only Hfq and full-length StpA display simultaneous binding of two RNAs, suggesting a matchmaker-like model for this activity. For StpA, this ''RNA crowding'' requires protein-protein interactions, since a dimerization-deficient StpA mutant lost the ability to bind and anneal two RNAs. These results underline the difference between the two reaction types, making it necessary to distinguish and classify proteins according to their specific RNA chaperone activities.

“…Also, after infection with coliphage QB, protein S1 becomes a subunit of the Qo replicase [ 3 ] . In vitro, the purified S1 is capable of binding to nuleic acids and unwinding a considerable amount of the residual secondary structure present in single-stranded DNA and RNA [4,5]. However, a mono-N-ethylmaleimide derivative of S1 which is readily incorporated into 30-S subunits deprived of S1, shows an almost complete loss of the helix-destabilizing activity [6,7].…”

mentioning

confidence: 99%

Nucleic Acid Binding and Unfolding Properties of Ribosomal Protein S1 and the Derivatives S1-F1 and m1-S1

et al. 1979

The nucleic acid binding and unwinding properties of wild-type Escherichiu coli ribosomal protein S1 have been compared to those of a mutant form and a large trypsin-resistant fragment, both reported recently [J. Mol. Biol. 127, 41 -45 (1979) and J . Biol. Chem. 254, 4309-4312 (1979). The mutant (ml-S1) contains 77% and the fragment (Sl-F1) 66% of the polypeptide chain length (z 600 amino acid residues) of protein S1. The mutant is active in protein synthesis in vitro; the fragment, although retaining one or more of the functional domains of S1, is inactive in protein synthesis. We find that ml-S1 is is almost as effective as S1 in binding to poly(rU), phage MS2 RNA and simian virus 40 (SV40) DNA, and in unfolding poly(rU) and the helical structures present in MS2 RNA and 4x174 viral DNA. S1-F1, however, binds to poly(rU) and denatured SV40 DNA, but not to MS2 RNA. It unfolds neither poly(rU), nor the residual secondary structure of MS2 RNA or 4x174 viral DNA. Thus, there appears to be a correlation between the loss in ability of S1 to unwind RNA and the loss in its ability to function in protein synthesis.Investigations from several laboratories have established an essential role for Escherichia coli ribosomal protein S1 in the binding of mRNA to ribosomes during the initiation of protein synthesis [l, 21. Also, after infection with coliphage QB, protein S1 becomes a subunit of the Qo replicase [ 3 ] . In vitro, the purified S1 is capable of binding to nuleic acids and unwinding a considerable amount of the residual secondary structure present in single-stranded DNA and RNA [4,5]. However, a mono-N-ethylmaleimide derivative of S1 which is readily incorporated into 30-S subunits deprived of S1, shows an almost complete loss of the helix-destabilizing activity [6,7]. This loss is accompanied by a decrease in the affinity for RNA and an inability to form an initiation complex with MS2 RNA [6,8].In order to gain further insight into the relationship between the nucleic acid binding and unwinding activities and the role of S1 in protein synthesis, we have compared the properties of S1 with those of two truncated derivatives: ml-S1 and S1-F1. Protein ml-S1 is a mutant from of S1 obtained from a phenotypically silent E. coli; it lacks about 140 amino acids from the C-terminal region, but Abbreviations. ml-S1 and S1-F1, a mutant form and a tryptic fragment of ribosomal protein S1; CD, circular dichroism; SV40, simian virus 40.functionally, is almost fully active in v i m [9] (and unpublished results of Subramanian). S1-F1 is a fragment of M , 48 500 produced by limited tryptic digestion of S1; it binds to poly(rU) and to 30-S subunits deprived of S1, but does not function in protein synthesis [lo]. The nucleic acid binding and unwinding properties of these two derivatives are reported in this paper. EXPERIMENTAL PROCEDURESRibosomal protein S1, the mutant protein (ml-S1) and the tryptic peptide from S1 (Sl-F1) were prepared as recently described [9--111. The slab gel electrophoretic pattern of the proteins used in the ...