Dataset Information

RNase Y, a novel endoribonuclease, initiates riboswitch turnover in Bacillus subtilis.

ABSTRACT: In contrast to Escherichia coli, initiation of mRNA decay in Gram-positive organisms is poorly understood. We studied the fate of the highly structured RNAs generated by premature transcription termination of S-adenosylmethionine (SAM)-dependent riboswitches in Bacillus subtilis. An essential protein of earlier unknown function, YmdA, was identified as a novel endoribonuclease (now called RNase Y) that was capable of preferential cleaving in vitro of the 5' monophosphorylated yitJ riboswitch upstream of the SAM-binding aptamer domain. Antiterminated full-length yitJ mRNA was not a substrate for RNase Y in vivo and in vitro, transcripts capable of forming the antiterminator were only cleaved in the presence of SAM. Turnover of 10 other SAM-dependent riboswitches was also initiated by RNase Y. Depletion of this ribonuclease increased the half-life of bulk mRNA more than two-fold. This indicates that RNase Y might be not only important for riboswitch RNA turnover but also as a key player in the initiation of mRNA decay in B. subtilis. About 40% of the sequenced eubacterial species have an RNase Y orthologue.

SUBMITTER: Shahbabian K

PROVIDER: S-EPMC2782095 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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RNase Y, a novel endoribonuclease, initiates riboswitch turnover in Bacillus subtilis.

Shahbabian Karen K Jamalli Ailar A Zig Léna L Putzer Harald H

The EMBO journal 20090924 22

In contrast to Escherichia coli, initiation of mRNA decay in Gram-positive organisms is poorly understood. We studied the fate of the highly structured RNAs generated by premature transcription termination of S-adenosylmethionine (SAM)-dependent riboswitches in Bacillus subtilis. An essential protein of earlier unknown function, YmdA, was identified as a novel endoribonuclease (now called RNase Y) that was capable of preferential cleaving in vitro of the 5' monophosphorylated yitJ riboswitch ups ...[more]

PMID: 19779461

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Project description:RNA-DNA hybrids are common in chromosomal DNA. Persistent RNA-DNA hybrids result in replication fork stress, DNA breaks, and neurological disorders in humans. During replication, Okazaki fragment synthesis relies on frequent RNA primer placement, providing one of the most prominent forms of covalent RNA-DNA strands in vivo The mechanism of Okazaki fragment maturation, which involves RNA removal and subsequent DNA replacement, in bacteria lacking RNase HI remains unclear. In this work, we reconstituted repair of a linear model Okazaki fragment in vitro using purified recombinant enzymes from Bacillus subtilis We showed that RNase HII and HIII are capable of incision on Okazaki fragments in vitro and that both enzymes show mild stimulation by single-stranded DNA binding protein (SSB). We also showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation in vitro Furthermore, we found that YpcP is a 5' to 3' nuclease that can act on a wide variety of RNA- and DNA-containing substrates and exhibits preference for degrading RNA in model Okazaki fragments. Together, our data showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation, whereas YpcP also contributes to the removal of RNA from an Okazaki fragment in vitroIMPORTANCE All cells are required to resolve the different types of RNA-DNA hybrids that form in vivo When RNA-DNA hybrids persist, cells experience an increase in mutation rate and problems with DNA replication. Okazaki fragment synthesis on the lagging strand requires an RNA primer to begin synthesis of each fragment. The mechanism of RNA removal from Okazaki fragments remains unknown in bacteria that lack RNase HI. We examined Okazaki fragment processing in vitro and found that RNase HIII in conjunction with DNA polymerase I represent the most efficient repair pathway. We also assessed the contribution of YpcP and found that YpcP is a 5' to 3' exonuclease that prefers RNA substrates with activity on Okazaki and flap substrates in vitro.

Dataset Information

RNase Y, a novel endoribonuclease, initiates riboswitch turnover in Bacillus subtilis.

Publications

RNase Y, a novel endoribonuclease, initiates riboswitch turnover in Bacillus subtilis.

Similar Datasets

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