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

Abstract

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 ...

Dynamics of Ribosomal Protein S1 on a Bacterial Ribosome with Cross-Linking and Mass Spectrometry

“…The localization of its individual domains reveals insights into the structure and interactions of the protein. The results are consistent with the notion that the N-terminal region of S1 binds the ribosome whereas the C-terminal region binds mRNA (10–13). S1 binds to the 30S subunit near the anti-Shine-Dalgarno motif at the 3′ end of the 16S rRNA, is highly elongated even when bound to the ribosome, and has a C-terminal RNA binding region with a cross-linking signature that suggests it is remarkably dynamic.…”

“…Analysis of fragments produced by limited proteolysis and chemical cleavage of S1 has shown that an N-terminal fragment of S1 (residues 1–193) binds the ribosome (10) but not RNA (11). Likewise, a C-terminal fragment (res 172–557) binds RNA (12, 13) but not the ribosome (6, 10). By nature of this bi-functional structure, S1 enhances the E. coli ribosome's affinity for RNA ∼5000 fold (14) and can directly mediate initiation of translation by binding the 5′ UTR of mRNA (4, 5).…”

Transfer-messenger RNA unfolds as it transits the ribosome

“…Unfolding of helix 5 and the abutting pseudoknots is likely to be facilitated by ribosomal protein S1 in Gramnegative and by RNA helicases in Gram-positive bacteria (Thomas et al 1979;Subramanian 1983;Kossen and Uhlenbeck 1999). Ribosomal protein S1 was shown to bind singlestranded and pseudoknotted RNA with high affinity (Bear et al 1976;Ringquist et al 1995).…”

Structural basis for RNA-genome recognition during bacteriophage Qβ replication

“…The S1 fragment, OB 1–2 , was found to bind primarily to the β-subunit of the Qβ replicase core complex, which complements early observations that the holoenzyme complex can dissociate into two entities: the EF-Tu–EF-Ts complex and a β-subunit·S1 complex ( 44 , 45 ). It is well established that the OB 1–2 domains of S1 are required for binding of the protein to both the ribosome and the β-subunit ( 14 , 23 , 31 – 33 ). Recent studies have narrowed this further down and identified the N-terminal 18 residues of OB 1 as essential for the interaction with the ribosome ( 23 ).…”

“…In particular, domains OB 1–3 were found to be essential for the recognition of a pseudoknot structure located in the ribosomal binding site of the E. coli rpsO mRNA ( 15 ). Additionally, an OB 1–2 -less S1 fragment was incapable of binding of a structured RNA and lacked the unwinding activity ( 33 ). Furthermore, a contribution of OB 1–2 towards the cooperative binding of mRNA and tmRNA by OB 3 has been observed ( 47 ).…”