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Guanosine pentaphosphate synthetase from Streptomyces antibioticus is also a polynucleotide phosphorylase.

ABSTRACT: The gene for the enzyme guanosine pentaphosphate synthetase I (GPSI) from Streptomyces antibioticus has been cloned and sequenced. The cloned gene functioned as a template in the streptomycete coupled transcription-translation system and directed the synthesis of a protein with the properties expected for GPSI. Sequencing of the cloned gene identified an open reading frame of 740 amino acids whose amino terminal sequence corresponded to the N terminus of purified GPSI. The GPSI protein sequence was found to possess significant homology to polynucleotide phosphorylase from Escherichia coli. Indeed, like E. coli polynucleotide phosphorylase, purified GPSI was shown to catalyze the polymerization of ADP and the phosphorolysis of poly(A). However, the E. coli enzyme was unable to catalyze the synthesis of guanosine pentaphosphate under conditions in which GPSI was highly active in that reaction. Overexpression of the cloned gpsI gene in E. coli led to an increase in both polynucleotide phosphorylase and guanosine pentaphosphate synthetase activities in the cloning host. The polynucleotide phosphorylase activities of GPSI and of the E. coli enzyme were strongly inhibited by dCDP, but the pppGpp synthetase activity of GPSI was not inhibited and indeed was slightly stimulated by dCDP. These results strongly support the identity of GPSI as a bifunctional enzyme capable of both pppGpp synthesis and polynucleotide phosphorylase activities.

SUBMITTER: Jones GH

PROVIDER: S-EPMC178187 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

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Guanosine pentaphosphate synthetase from Streptomyces antibioticus is also a polynucleotide phosphorylase.

Jones G H GH Bibb M J MJ

Journal of bacteriology 19960701 14

The gene for the enzyme guanosine pentaphosphate synthetase I (GPSI) from Streptomyces antibioticus has been cloned and sequenced. The cloned gene functioned as a template in the streptomycete coupled transcription-translation system and directed the synthesis of a protein with the properties expected for GPSI. Sequencing of the cloned gene identified an open reading frame of 740 amino acids whose amino terminal sequence corresponded to the N terminus of purified GPSI. The GPSI protein sequence ...[more]

PMID: 8763958

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Project description:We have examined the expression of pnp encoding the 3'-5'-exoribonuclease, polynucleotide phosphorylase, in Streptomyces antibioticus. We show that the rpsO-pnp operon is transcribed from at least two promoters, the first producing a readthrough transcript that includes both pnp and the gene for ribosomal protein S15 (rpsO) and a second, Ppnp, located in the rpsO-pnp intergenic region. Unlike the situation in Escherichia coli, where observation of the readthrough transcript requires mutants lacking RNase III, we detect readthrough transcripts in wild-type S. antibioticus mycelia. The Ppnp transcriptional start point was mapped by primer extension and confirmed by RNA ligase-mediated reverse transcription-PCR, a technique which discriminates between 5' ends created by transcription initiation and those produced by posttranscriptional processing. Promoter probe analysis demonstrated the presence of a functional promoter in the intergenic region. The Ppnp sequence is similar to a group of promoters recognized by the extracytoplasmic function sigma factors, sigma-R and sigma-E. We note a number of other differences in rspO-pnp structure and function between S. antibioticus and E. coli. In E. coli, pnp autoregulation and cold shock adaptation are dependent upon RNase III cleavage of an rpsO-pnp intergenic hairpin. Computer modeling of the secondary structure of the S. antibioticus readthrough transcript predicts a stem-loop structure analogous to that in E. coli. However, our analysis suggests that while the readthrough transcript observed in S. antibioticus may be processed by an RNase III-like activity, transcripts originating from Ppnp are not. Furthermore, the S. antibioticus rpsO-pnp intergenic region contains two open reading frames. The larger of these, orfA, may be a pseudogene. The smaller open reading frame, orfX, also observed in Streptomyces coelicolor and Streptomyces avermitilis, may be translationally coupled to pnp and the gene downstream from pnp, a putative protease.

Dataset Information

Guanosine pentaphosphate synthetase from Streptomyces antibioticus is also a polynucleotide phosphorylase.

Publications

Guanosine pentaphosphate synthetase from Streptomyces antibioticus is also a polynucleotide phosphorylase.

Similar Datasets

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