Messenger RNA Recognition by Fragments of Ribosomal Protein S4

Anne-Marie Baker,David E Draper

doi:10.1074/jbc.270.39.22939

Anne-Marie Baker, David E Draper

Open Access

https://doi.org/10.1074/jbc.270.39.22939

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Abstract

Ribosomal protein S4 from Escherichia coli binds a large domain of 16 S ribosomal RNA and also a pseudoknot structure in the alpha operon mRNA, where it represses its own synthesis. No similarity between the two RNA binding sites has been detected. To find out whether separate protein regions are responsible for rRNA and mRNA recognition, proteins with N-terminal or C-terminal deletions have been overexpressed and purified. Protein-mRNA interactions were detected by (i) a nitrocellulose filter binding assay, (ii) inhibition of primer extension by reverse transcriptase, and (iii) a gel shift assay. Circular dichroism spectra were taken to determine whether the proteins adopted stable secondary structures. From these studies it is concluded that amino acids 48-104 make specific contacts with the mRNA, although residues 105-177 (out of 205) are required to observe the same toeprint pattern as full-length protein and may stabilize a specific portion of the mRNA structure. These results parallel ribosomal RNA binding properties of similar fragments (Conrad, R. C., and Craven, G. R. (1987) Nucleic Acids Res. 15, 10331-10343, and references therein). It appears that the same protein domain is responsible for both mRNA and rRNA binding activities.

Highlights

In several instances there is convincing similarity between the secondary structures of the mRNA and rRNA targets of an autoregulatory ribosomal protein (4 – 6)
The intact E. coli S4 protein was expressed by this method, and could be purified in higher yield (ϳ40 mg of protein/liter of cell culture; see “Materials and Methods”) and with greater ease than possible by standard methods that rely on extraction of protein from purified ribosomes or ribosome subunits [23, 27]
Comparison of 16 S rRNA and ␣ mRNA Binding by S4 Fragments—N- and C-terminal deletions of S4, similar to the ones described in this work, were prepared by Craven and colleagues and tested for binding to 16 S ribosomal RNA and, in some cases, for ribosome assembly

Summary

Introduction

In several instances there is convincing similarity between the secondary structures of the mRNA and rRNA targets of an autoregulatory ribosomal protein (4 – 6). The entire 5Ј domain of the 16 S rRNA, a fragment of 460 nucleotides, is needed to form the ribosomal binding site for the protein [8], a smaller region is protected from cleavage by bound protein [9]. We have prepared a number of truncated proteins by overexpression of the S4 gene rather than cleavage methods, and show that Nand C-terminal regions of S4 that are not necessary for rRNA binding are not required for S4 recognition of the ␣ mRNA pseudoknot. The results show that no more than ϳ130 of the 205 amino acids are needed to fold a stable RNA binding domain, and suggest that two regions within this domain may recognize different parts of the RNA

Methods

Results

Conclusion