A pRNA‐induced structural rearrangement triggers 6S‐1 RNA release from RNA polymerase in Bacillus subtilis

Bacillus subtilis 6S‐1 RNA binds to the housekeeping RNA polymerase (σA‐RNAP) and directs transcription of short ‘product’ RNAs (pRNAs). Here, we demonstrate that once newly synthesized pRNAs form a sufficiently stable duplex with 6S‐1 RNA, a structural rearrangement is induced in cis, which involves base‐pairing between sequences in the 5′‐portion of the central bulge and nucleotides that become available as a result of pRNA invasion. The rearrangement decreases 6S‐1 RNA affinity for σA‐RNAP. Among the pRNA length variants synthesized by σA‐RNAP (up to ∼14 nt), only the longer ones, such as 12–14‐mers, form a duplex with 6S‐1 RNA that is sufficiently long‐lived to induce the rearrangement. Yet, an LNA (locked nucleic acid) 8‐mer can induce the same rearrangement due to conferring increased duplex stability. We propose that an interplay of rate constants for polymerization (kpol), for pRNA:6S‐1 RNA hybrid duplex dissociation (koff) and for the rearrangement (kconf) determines whether pRNAs dissociate or rearrange 6S‐1 structure to trigger 6S‐1 RNA release from σA‐RNAP. A bioinformatic screen suggests that essentially all bacterial 6S RNAs have the potential to undergo a pRNA‐induced structural rearrangement.

[1]  Terence R. Strick,et al.  Abortive Initiation and Productive Initiation by RNA Polymerase Involve DNA Scrunching , 2006, Science.

[2]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[3]  G. Storz,et al.  6S RNA Regulates E. coli RNA Polymerase Activity , 2000, Cell.

[4]  L. Hsu.  Monitoring abortive initiation. , 2009, Methods.

[5]  K. Wassarman,et al.  Synthesis-Mediated Release of a Small RNA Inhibitor of RNA Polymerase , 2006, Science.

[6]  E. Viñuela,et al.  RNA polymerase binding sites and transcription map of the DNA of Bacillus subtilis phage phi29. , 1979, Journal of molecular biology.

[7]  R. Wagner,et al.  6S RNA-dependent inhibition of RNA polymerase is released by RNA-dependent synthesis of small de novo products , 2010, Biological chemistry.

[8]  J. Wengel,et al.  LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. , 2004, Biochemistry.

[9]  Harald Schwalbe,et al.  Time-resolved NMR methods resolving ligand-induced RNA folding at atomic resolution. , 2007, Proceedings of the National Academy of Sciences of the United States of America.

[10]  W. L. Ruzzo,et al.  6S RNA is a widespread regulator of eubacterial RNA polymerase that resembles an open promoter. , 2005, RNA.

[11]  R. Wagner,et al.  Studies on the function of the riboregulator 6S RNA from E. coli: RNA polymerase binding, inhibition of in vitro transcription and synthesis of RNA-directed de novo transcripts , 2007, Nucleic acids research.

[12]  G. Brownlee.  Sequence of 6S RNA of E. coli. , 1971, Nature: New biology.

[13]  R. Hartmann,et al.  Locked Nucleic Acid Oligonucleotides , 2012, BioDrugs.

[14]  Lindsay Shephard,et al.  Binding and release of the 6S transcriptional control RNA. , 2010, RNA.

[15]  B. Beckmann,et al.  In vivo and in vitro analysis of 6S RNA-templated short transcripts in Bacillus subtilis , 2011, RNA biology.

[16]  K. Yamane,et al.  Expression of a small RNA, BS203 RNA, from the yocI-yocJ intergenic region of Bacillus subtilis genome. , 2002, FEMS microbiology letters.

[17]  R. Hartmann,et al.  Locked nucleic acid oligonucleotides: the next generation of antisense agents? , 2007, BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy.

[18]  G. Storz,et al.  Regulatory RNAs in Bacteria , 2009, Cell.

[19]  Shimon Weiss,et al.  Initial Transcription by RNA Polymerase Proceeds Through a DNA-Scrunching Mechanism , 2006, Science.

[20]  K. Wassarman,et al.  A highly conserved 6S RNA structure is required for regulation of transcription , 2005, Nature Structural &Molecular Biology.

[21]  K. Wassarman.  6S RNA: a regulator of transcription , 2007, Molecular microbiology.

[22]  Kristin Reiche,et al.  The primary transcriptome of the major human pathogen Helicobacter pylori , 2010, Nature.

[23]  B. Beckmann,et al.  Northern blot detection of endogenous small RNAs (∼14 nt) in bacterial total RNA extracts , 2010, Nucleic acids research.

[24]  Roland K. Hartmann,et al.  6S RNA – an ancient regulator of bacterial RNA polymerase rediscovered , 2005, Biological chemistry.

[25]  Robert D. Finn,et al.  Rfam: updates to the RNA families database , 2008, Nucleic Acids Res..

[26]  A. Serganov.  The long and the short of riboswitches. , 2009, Current opinion in structural biology.

[27]  Peter F. Stadler,et al.  ViennaRNA Package 2.0 , 2011, Algorithms for Molecular Biology.

[28]  K. Bunai,et al.  Identification and characterization of novel small RNAs in the aspS-yrvM intergenic region of the Bacillus subtilis genome. , 2002, Microbiology.