Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria.

Autor:	Woods D; Department of Biological Sciences, University at Albany, Albany, New York, USA., Vangaveti S; The RNA Institute, University at Albany, Albany, New York, USA., Egbanum I; Department of Biological Sciences, University at Albany, Albany, New York, USA., Sweeney AM; Department of Biology, Murray State University, Murray, Kentucky, USA., Li Z; Wadsworth Center, New York State Department of Health, Albany, New York, USA., Bacot-Davis V; Renaissance School of Medicine, Stony Brook University, Stony Brook, New York, USA., LeSassier DS; Signature Science, LLC, Austin, Texas, USA., Stanger M; Department of Biological Sciences, University at Albany, Albany, New York, USA., Hardison GE; Department of Biology, Murray State University, Murray, Kentucky, USA., Li H; Department of Biological Sciences, University at Albany, Albany, New York, USA.; Wadsworth Center, New York State Department of Health, Albany, New York, USA., Belfort M; Department of Biological Sciences, University at Albany, Albany, New York, USA mbelfort@albany.edu clennon1@murraystate.edu.; The RNA Institute, University at Albany, Albany, New York, USA., Lennon CW; Department of Biology, Murray State University, Murray, Kentucky, USA mbelfort@albany.edu clennon1@murraystate.edu.
Jazyk:	angličtina
Zdroj:	MBio [mBio] 2020 Jul 14; Vol. 11 (4). Date of Electronic Publication: 2020 Jul 14.
DOI:	10.1128/mBio.01403-20
Abstrakt:	Inteins, as posttranslational regulatory elements, can tune protein function to environmental changes by conditional protein splicing (CPS). Translated as subdomains interrupting host proteins, inteins splice to scarlessly join flanking sequences (exteins). We used DnaB-intein1 (DnaBi1) from a replicative helicase of Mycobacterium smegmatis to build a k anamycin i ntein s plicing r eporter (KISR) that links splicing of DnaBi1 to kanamycin resistance. Using expression in heterologous Escherichia coli , we observed phenotypic classes of various levels of splicing-dependent resistance (SDR) and related these to the insertion position of DnaBi1 within the kanamycin resistance protein (KanR). The KanR-DnaBi1 construct demonstrating the most stringent SDR was used to probe for CPS of DnaB in the native host environment, M. smegmatis We show here that zinc, important during mycobacterial pathogenesis, inhibits DnaB splicing in M. smegmatis Using an in vitro reporter system, we demonstrated that zinc potently and reversibly inhibited DnaBi1 splicing, as well as splicing of a comparable intein from Mycobacterium leprae Finally, in a 1.95 Å crystal structure, we show that zinc inhibits splicing through binding to the very cysteine that initiates the splicing reaction. Together, our results provide compelling support for a model whereby mycobacterial DnaB protein splicing, and thus DNA replication, is responsive to environmental zinc. IMPORTANCE Inteins are present in a large fraction of prokaryotes and localize within conserved proteins, including the mycobacterial replicative helicase DnaB. In addition to their extensive protein engineering applications, inteins have emerged as environmentally responsive posttranslational regulators of the genes that encode them. While several studies have shown compelling evidence of conditional protein splicing (CPS), examination of splicing in the native host of the intein has proven to be challenging. Here, we demonstrated through a number of measures, including the use of a splicing-dependent sensor capable of monitoring intein activity in the native host, that zinc is a potent and reversible inhibitor of mycobacterial DnaB splicing. This work also expands our knowledge of site selection for intein insertion within nonnative proteins, demonstrating that splicing-dependent host protein activation correlates with proximity to the active site. Additionally, we surmise that splicing regulation by zinc has mycobacteriocidal and CPS application potential. (Copyright © 2020 Woods et al.)
Databáze:	MEDLINE
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