The unusually long amino acid acceptor stem of<i>Escherichia coli</i>selenocysteine tRNA results from abnormal cleavage by RNase P

A detailed analysis of the expression of the sel genes, the products of which are necessary for the specific incorporation of selenium into macromolecules in Escherichia coli, showed that transcription was constitutive, being influenced neither by atrobiosis or anaerobiosis nor by the intracellular selenium concentration. The gene encoding the tRNA molecule which is specifically aminoacylated with selenocysteine (seiC) proved to be monocistronic. In contrast, the other three sel genes (seL4, -B, and -D) were shown to be constituents of two unlinked operons. The selA and selB geites formed one transcriptional unit (seL41)7, while selD was shown to be the central gene in an operon including two other genes, the promoter distal of which (topB) encodes topoisomerase HI. The promoter proximal gene (orfl83) was sequenced and shown to encode a protein consisting of 183 amino acids (Mr,20,059), the amino acid sequence of which revealed no similarity to any currently known protein. The products of the orfl83 and topB genes were required neither for selenoprotein biosynthesis nor for selenation of tRNAs. seL4B transcription was driven by a single, weak promoter; however, two major selD operon transcripts were identified. The longer initiated just upstream of the orfl83 gene, whereas the 5' end of the other mapped in a 116-bp nontranslated region between orfl83 and selD. Aerobic synthesis of all four sel gene products incited a reexamination of a weak 110-kDa selenopolypeptide which is produced under these conditions. The aerobic appearance of this 110-kDa selenopolypeptide was not a consequence of residual expression of the gene encoding the 110-kDa selenopolypeptide of the anaerobically inducible formate dehydrogenase N (FDHN) enzyme, as previously surmised, but rather resulted from the expression of a gene encoding a third, distinct selenopolypeptide in E. coli. A mutant strain no longer capable of synthesizing the 80-and 110-kDa selenopolypeptides of FDHH and FDHN, respectively, still synthesized this alternative 110-kDa selenopolypeptide which was present at equivalent levels in cells grown aerobically and anaerobically with nitrate. Furthermore, this strain exhibited a formate-and sel gene-dependent respiratory activity, indicating that it is probable that this selenopolypeptide constitutes a major component of the formate oxidase, an enzyme activity initially discovered in aerobically grown E. coli more than 30 years ago.The products of four genes have been identified as being essential for the incorporation of selenium into macromolecules in Escherichia coli (8,44). The selD gene encodes a protein which is required for incorporation of selenium into both protein and modified tRNAs (28), whereas the products of the other three genes are required for the synthesis of selenopolypeptides (29). The selC gene product is a tRNA (30) which is aminoacylated with serine, and this serine residue is converted to selenocysteine on the tRNA through the joint action of SelD (28) and selenocysteine synthase, the selA gene product ...

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“…The migrated more slowly in the gel were observed. These results are in agreement with the findings of Burkard and Soll (9).…”

Section: Resultssupporting

confidence: 94%

“…4B) analysis. That of the selC gene has been determined previously (9). RNA was isolated from aerobically grown cells and was hybridized with radioactively labelled oligonucleotides which were predicted to prime between 100 and 140 bases downstream of the respective initiation sites, on the basis of the results presented in Fig.…”

Section: Resultsmentioning

confidence: 99%

Expression and operon structure of the sel genes of Escherichia coli and identification of a third selenium-containing formate dehydrogenase isoenzyme

Sawers¹,

Heider²,

Zehelein³

et al. 1991

J Bacteriol

115

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“…This structure contains a quasi-continuous helix that closely approximates the stacked AA and T arms of a normal tRNA, even though it includes a pseudoknot [7]. Other studies indicate that site selection by RNase P is critically dependent on the length of(i.e., number of base pairs in) the AA stem [1][2][3]5]. In this connection, it is noteworthy that the t-elements described here cannot form distal base pairs in the AA (t3) or T (t2) arms, or both (tl), while at the shme time they have an AA stem which could be extended by an additional one (t2) or three (t~) base pairs at the -C C A o H end.…”

Section: Discussionmentioning

confidence: 99%

In vitro processing of transcripts containing novel tRNA-like sequences (?t-elements?) encoded by wheat mitochondrial DNA

1990

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We have recently described the properties of a wheat mitochondrial extract that is able to process, accurately and efficiently, artificial transcripts containing wheat mitochondrial tRNA sequences, with the production of mature tRNAs (P.J. Hanic-Joyce and M.W. Gray, J. Biol. Chem., in press). Such processing involves 5'-endonucleolytic, 3'-endonucleolytic, and tRNA nucleotidyltransferase activities. Here we show that this system also acts on transcripts containing sequences corresponding to an unusual class of short repeats ('t-elements') in wheat mtDNA. These repeats are theoretically capable of assuming a tRNA-like secondary structure, although stable transcripts corresponding to them are not detectable in vivo. We find that t-element sequences are processed with the same specificity and with comparable efficiency as are authentic tRNA sequences. Because known t-elements are located close to and in the same transcriptional orientation as active genes (18S-5S, 26S, tRNA(Pro)) in wheat mtDNA, our results raise the question of whether t-elements play a role in gene expression in wheat mitochondria.

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“…15 Previous work using various tRNA precursors indicated the importance of guanosine at the cleavage site with respect to cleavage site recognition and efficiency of cleavage under physiological pH conditions. 4,5,7,8,[26][27][28] Moreover, the influence of the presence of G C1 /C C72 on substrate binding and rate of cleavage has been documented both for E. coli and Bacillus subtilis RNase P. 2,10 Based on data where we studied precursors where G C1 /C C72 had been replaced with U C1 /A C72 we recently concluded that the C1/C72 base-pair in a tRNA precursor plays an important role in substrate discrimination by RNase RNA. 2 In the present study we showed that replacement/deletion of chemical groups of G C1 at the RNase P cleavage site affected cleavage site recognition, ground state binding and rate of cleavage under conditions where chemistry is suggested to be rate-limiting.…”

Section: Influence Of the 2 0 Oh And N7 Of G C1 On Catalysismentioning

confidence: 99%

The Exocyclic Amine at the RNase P Cleavage Site Contributes to Substrate Binding and Catalysis

Kikovska

Brännvall

Kirsebom

2006

Journal of Molecular Biology

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The unusually long amino acid acceptor stem ofEscherichia coliselenocysteine tRNA results from abnormal cleavage by RNase P

Cited by 30 publications

References 13 publications

Expression and operon structure of the sel genes of Escherichia coli and identification of a third selenium-containing formate dehydrogenase isoenzyme

Expression and operon structure of the sel genes of Escherichia coli and identification of a third selenium-containing formate dehydrogenase isoenzyme

In vitro processing of transcripts containing novel tRNA-like sequences (?t-elements?) encoded by wheat mitochondrial DNA

The Exocyclic Amine at the RNase P Cleavage Site Contributes to Substrate Binding and Catalysis

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