Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3′-to-5′ Helicase That Unwinds 3′-Tailed RNA Duplexes and RNA:DNA Hybrids

Abstract

Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3= processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3=-to-5= helicase. HelY requires a 3= single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3=-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. H elicases are nucleic acid-dependent nucleoside triphosphatases (NTPases) that play important roles in diverse nucleic acid transactions. DNA helicases orchestrate replication, recombination, and repair. RNA helicases choreograph transcription, RNA processing, ribosome biogenesis, translation, and RNA turnover. Helicases use the chemical energy of nucleoside triphosphate (NTP) hydrolysis either to effect mechanical changes in the secondary structure of nucleic acids or to remodel (or disrupt) the structures of protein-nucleic acid complexes. Helicases are classified into superfamilies, families, and subfamilies according to their distinctive primary, tertiary, and quaternary structures and their biochemical specificities, i.e., NTP preference, nucleic acid preference (duplex DNA, duplex RNA, or RNA:DNA hybrids), and directionality of translocation or unwinding (5= to 3= or 3= to 5=) (1).Differences in the roster of helicases between taxa offer useful clues to the evolution and diversification of DNA replication/repair and RNA synthesis/processing strategies. Where the helicase rosters diverge in animals versus pathogens, they can suggest antiinfective drug targets. With this in mind, we are focused on the helicases of members of the genus Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative, M. smegmatis. To date, we have purified and characterized six mycobacterial DNA helicases: AdnAB (2, 3, 4), UvrD1 (5, 6), UvrD2 (7), SftH (8), RqlH (9), and Lhr (10). Mycobacterial DNA helicases XBP (11, 12), RecG (13,14,15), RecD (16), RuvB (17), and DnaB (18) have been purified and characterized by other investigators.Comparatively little attention has been paid to the mycobacterial RNA helicase repertoire. The transcription termination factor Rho is, to our knowledge, the lone example of a biochemically characterized NTP-driven RNA translocase/helicase from a Mycobacterium spec...

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Genome-Wide Transcriptional Response of Mycobacterium smegmatis MC2155 to G-Quadruplex Ligands BRACO-19 and TMPyP4

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