Escherichia coli ribosomal protein S1 has two polynucleotide binding sites.

Abstract

The interaction of Escherichia coli ribosomal protein SI with a variety of RNA and DNA oligomers and polymers has been studied, using both a sedimentation technique and the quenching of intrinsic protein fluorescence upon nucleic acid binding to obtain equilibrium binding parameters. Two polynucleotide binding sites have been detected on SI: site I binds either single-stranded DNA or RNA and does not discriminate between adenine-and cytidine-containing polynucleotides, while site II binding is highly specific for RNA over DNA and shows a marked preference for cytidine polynucleotides over the corresponding adenine-containing species. On the basis of the binding properties of SI to denatured DNA cellulose and poly(rC)cellulose, it is demonstrated that every SI molecule carries both a site I and a site II. Some possible implications of these results for mechanisms of protein synthesis and phage Qft replication are briefly considered.The Escherichia coli ribosomal protein SI has a number of properties which suggest that it plays a very central role in protein synthesis and RNA phage replication. Although it is a "fractional" protein [i.e., it is present in less than one copy per purified 30S subunit (1)], it is virtually required for the binding and translation of poly(rU) and some natural messenger RNAs (2). Si has also been shown to be the "factor i" protein (3, 4) described by Revel et al. (5), which inhibits translation in in vitro systems at molar ratios of protein to ribosomes greater than -one (6). This inhibition is apparently due to Si binding the mRNA directly (7) and is most effective with polypyrimidine messengers and least effective with polypurines (8). Further, the phage Qf3 codes for a replicase that complexes with Si and two other host proteins (3). S1 is required in this complex for recognition and replication of the phage plus strand, but not the complementary minus strand (9).All three of these activities of Si-binding mRNA to the ribosome, inhibiting translation, and aiding replication of Qf RNA-probably depend in some way on the ability of SI to bind polynucleotides. However, the mechanisms by which Si functions in any of these three activities are not really understood in any detail. With regard to the influence of Si on the binding and translation of mRNA, a number of suggestions have been made: (i) that SI forms part of the mRNA binding site on the ribosome (10); (ii) that Si melts a region of secondary structure in the 16S ribosomal RNA to facilitate formation of base pairs between the rRNA and mRNA in initiation of translation (11); (iii) that Si acts to stabilize this same basepaired region (12); and (iv) that Si affects the tertiary structure of mRNA to allow it to bind the 30S subunit (13).Although these hypotheses predict different nucleic acid binding properties for Si, information on the interaction of this protein with nucleic acids is meager. The ability of Si to bindThe costs of publication of this article were defrayed in part by the payment of page charges. This articl...

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

“…Other models of cooperativity are possible but seem less likely. For instance, binding of one protein could expose a second, stronger binding site in the RNA (28), but this same event would have to occur on tmRNA and our model mRNA, which differ dramatically in sequence and structure.…”

Interaction of Escherichia coli ribosomal protein S1 with ribosomes.

“…For this system we assume that equilibration is fast relative to the rate of sedimentation (the time required to sediment the ribosomal subunit peak by one bandwidth is greater than 10 Tables 1 and 2); the latter determinations are based on fluorescence measurements made at equilibrium under "nontransport" conditions (5,6 still cosedimenting with the ligand at the end of the experiment is thus'generally not the fraction bound prior to sedimentation (as is frequently, and erroneously, assumed); rather, a much smaller amount of protein remains bound and the experiment should be viewed as analogous to the multiple and sequential equilibria that apply in column chromatography, in which free protein is progressively washed out of the complex (i.e., off the column) by successive aliquots of buffer. Given the distance of sedimentation and original thickness of the band, the fraction of binding protein still cosedimenting with the fast component, and the concentration of binding sites on the rapidly sedimenting component, an association constant can be calculated (12) (4). Thus, by setting up competitive binding experiments in which Si is partitioned between 30S subunits and various polynucleotides, we can determine which (if either) of the polynucleotide binding sites of Si is involved in the interaction with 30S ribosomal subunits (presumably with exposed portions of the 16S rRNA) and which sites might remain available for binding of mRNA or other nucleic acid components.…”

“…Polyribonucleotides were purchased from Miles; polydeoxyribonucleotides were from Collaborative Research (Waltham, MA). S1 protein labeled with [3H]leucine (New England Nuclear) and unlabeled S1 were prepared from E. coli MRE 600 cells by DNA-cellulose chromatography as described (4,5).t [3H]S1 and unlabeled S1 coelectrophoresed in sodium dodecyl sulfate/polyacrylamide gels and competed for binding to the 30S ribosomal subunit. "High-salt-washed" 30S ribosomal subunits and 70S ribosomes were prepared from E. coli MRE (1979) 1041 600 cells by standard procedures (I1), and 30S subunits more than 98% depleted of SI protein were prepared by a low.-salt treatment (3).…”

“…in the activated state. Concentrations of SI and of ribosomal particles were calculated by using published extinction coefficients (4,11).…”

“…Recently, we demonstrated the presence of two distinct polynucleotide binding sites on Si protein (4). Site I binds well to single-stranded RNA or DNA, interacts mostly with the sugar-phosphate backbone, and shows little dependence of binding on base composition (5).…”