Specific ribosomal RNA recognition by a fragment of<i>E. coli</i>ribosomal protein S4 missing the C-terminal 36 amino acid residues

Changchien, Li-Ming; Craven, Gary R.

doi:10.1093/nar/13.17.6343

Cited by 7 publications

(11 citation statements)

References 29 publications

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“…Fragments terminating at residues 171, 119 and 101 ( B.stearothermophilus numbering) were also made. All retained some affinity for rRNA (Changchien and Craven, 1985, 1986; Conrad and Craven, 1987). However, later studies using circular dichroism showed that any truncation of the molecule prior to residue 171 resulted in a marked destabilization (Baker and Draper, 1995).…”

Section: Discussionmentioning

confidence: 99%

The crystal structure of ribosomal protein S4 reveals a two-domain molecule with an extensive RNA-binding surface: one domain shows structural homology to the ETS DNA-binding motif

et al. 1998

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We report the 1.7 Å crystal structure of ribosomal protein S4 from Bacillus stearothermophilus. To facilitate the crystallization, 41 apparently flexible residues at the N-terminus of the protein have been deleted (S4Δ41). S4Δ41 has two domains; domain 1 is completely α-helical and domain 2 comprises a fivestranded antiparallel β-sheet with three α-helices packed on one side. Domain 2 is an insertion within domain 1, and it shows significant structural homology to the ETS domain of eukaryotic transcription factors. A phylogenetic analysis of the S4 primary structure shows that the likely RNA interaction surface is predominantly on one side of the protein. The surface is extensive and highly positively charged, and is centered on a distinctive canyon at the domain interface. The latter feature contains two arginines that are totally conserved in all known species of S4 including eukaryotes, and are probably crucial in binding RNA. As has been shown for other ribosomal proteins, mutations within S4 that affect ribosome function appear to disrupt the RNA-binding sites. The structure provides a framework with which to probe the RNA-binding properties of S4 by site-directed mutagenesis.

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Section: Discussionmentioning

confidence: 99%

The crystal structure of ribosomal protein S4 reveals a two-domain molecule with an extensive RNA-binding surface: one domain shows structural homology to the ETS DNA-binding motif

et al. 1998

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“…S4 fragments with C-terminal deletions produced by Me 2 SOHBr reaction with Trp-170, hydroxylamine cleavage at Asn-123/Gly-124, or cyanogen bromide reaction at Met-105 have been described (14,15,41). All of these interact preferentially with 16 S rRNA over 23 S rRNA in an assay in which the binding of labeled protein to nitrocellulose filters is prevented by formation of the protein-RNA complex (42).…”

Section: Mrna Binding Of Ribosomal Protein S4 Fragmentsmentioning

confidence: 99%

“…In this paper we build on the previous work of Craven and colleagues (12)(13)(14)(15), who cleaved S4 by various methods and studied the ability of the protein fragments to bind 16 S rRNA and promote ribosome assembly in vitro. We have prepared a number of similarly 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 also not required for S4 recognition of the ␣ mRNA pseudoknot.…”

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confidence: 99%

Messenger RNA Recognition by Fragments of Ribosomal Protein S4

Baker

Draper

1995

Journal of Biological Chemistry

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Ribosomal protein S4 from Escherichia coli binds a large domain of 16 S ribosomal RNA and also a pseudoknot structure in the ␣ 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 fulllength 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.Functional studies of ribosomes have tended to focus on the roles of the ribosomal RNAs in recent years, as a number of studies have uncovered specific contributions of different rRNA domains to ribosome activities (1). As more ribosomal protein sequences have become available, it is becoming clear that a number of these proteins are highly conserved among all organisms and must also have specific and necessary roles in ribosome function. An intriguing set of ribosomal proteins are those that bind directly and independently to the ribosomal RNAs and also autogeneously regulate ribosomal protein expression. In many cases the regulation is due to the protein recognition of the mRNA translational initiation region (2), although protein binding to a pre-mRNA splice site has also been observed (3). These instances of a single protein carrying out two different RNA-related functions provide interesting systems for studying how RNA recognition has evolved and is related to specific protein functions (4).In several instances there is convincing similarity between the secondary structures of the mRNA and rRNA targets of an autoregulatory ribosomal protein (4 -6). It is reasonable to conclude that both mRNA and rRNA bind in the same active site of any one of these proteins. In other cases there is no obvious similarity between the two RNA substrates. For instance, the mRNA target site for Escherichia coli S4 protein is a complex pseudoknot of about 110 nucleotides within the ␣ operon mRNA (7). Nearly 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), although a smaller region is protected from cleavage by bound protein (9). There is no primary or secondary structural similarity between the two target sites, ...

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“…We have determined that the N-terminal 46 amino acids of S4 are not required for S4 binding to 16S RNA, although residues 31-46 are involved in assembly (6). We have also shown that residues 168-203 are not involved in rRNA binding (7); they seem however to be involved in conformational changes in assembly and in the translational repression of the S4 operon (8,9). The smallest active fragment of S4 that we have generated so far consisted of residues 1-124 and was produced by hydroxylamine cleavage (10).…”

mentioning

confidence: 90%

“…We report here the production of a fragment of S4 consisting of residues 1-103, generated by cyanogen bromide (CNBr) cleavage, which rebinds 16S RNA with an affinity comparable to intact S4. However, this loss of almost half of the protein has severely affected its ability to participate in 30S assembly, not so much in terms of protein incorporation, but in a conformational contraction proposed to take place at a late stage in assembly (7).…”

mentioning

confidence: 99%

A cyanogen bromide fragment of S4 that specifically rebinds 16S RNA

Conrad

Craven

1987

Nucl Acids Res

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Escherichia coli ribosomal protein S4 was subjected to cyanogen bromide cleavage and was found to generate a complete cleavage product capable of rebinding 16S rRNA. This fragment, consisting of residues 1-103, was found to bind with an apparent association constant of 11 microM-1. This fragment was used in place of S4 in an in vitro reconstitution experiment. Although the particles formed had a protein composition not significantly different from reconstituted 30S ribosomal subunits, their sedimentation behavior was more like that of particles reconstituted without S4. These results indicate to us that, although residues 104-203 of S4 are involved in the assembly of the 30S ribosome, they are not necessary for the binding of S4 to 16S RNA. Taken with previous results, the domain of S4 involved in specific binding of 16S RNA can be confined to residues 47-103.

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Specific ribosomal RNA recognition by a fragment ofE. coliribosomal protein S4 missing the C-terminal 36 amino acid residues

Cited by 7 publications

References 29 publications

The crystal structure of ribosomal protein S4 reveals a two-domain molecule with an extensive RNA-binding surface: one domain shows structural homology to the ETS DNA-binding motif

The crystal structure of ribosomal protein S4 reveals a two-domain molecule with an extensive RNA-binding surface: one domain shows structural homology to the ETS DNA-binding motif

Messenger RNA Recognition by Fragments of Ribosomal Protein S4

A cyanogen bromide fragment of S4 that specifically rebinds 16S RNA

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