Mechanistic comparison of Bacillus subtilis 6S-1 and 6S-2 RNAs--commonalities and differences.
Bacterial 6S RNAs bind to the housekeeping RNA polymerase (σ(A)-RNAP in Bacillus subtilis) to regulate transcription in a growth phase-dependent manner. B. subtilis expresses two 6S RNAs, 6S-1 and 6S-2 RNA, with different expression profiles. We show in vitro that 6S-2 RNA shares hallmark features with 6S-1 RNA: Both (1) are able to serve as templates for pRNA transcription; (2) bind with comparable affinity to σ(A)-RNAP; (3) are able to specifically inhibit transcription from DNA promoters, and (4) can form stable 6S RNA:pRNA hybrid structures that (5) abolish binding to σ(A)-RNAP. However, pRNAs of equal length dissociate faster from 6S-2 than 6S-1 RNA, owing to the higher A,U-content of 6S-2 pRNAs. This could have two mechanistic implications: (1) Short 6S-2 pRNAs (<10 nt) dissociate faster instead of being elongated to longer pRNAs, which could make it more difficult for 6S-2 RNA-stalled RNAP molecules to escape from the sequestration; and (2) relative to 6S-1 RNA, 6S-2 pRNAs of equal length will dissociate more rapidly from 6S-2 RNA after RNAP release, which could affect pRNA turnover or the kinetics of 6S-2 RNA binding to a new RNAP molecule. As 6S-2 pRNAs have not yet been detected in vivo, we considered that cellular RNAP release from 6S-2 RNA might occur via 6S-1 RNA displacing 6S-2 RNA from the enzyme, either in the absence of pRNA transcription or upon synthesis of very short 6S-2 pRNAs (∼ 5-mers, which would escape detection by deep sequencing). However, binding competition experiments argued against these possibilities.
Olga Y
OY
Burenina
Philipp G
PG
Hoch
Katrin
K
Damm
Margarita
M
Salas
Timofei S
TS
Zatsepin
Marcus
M
Lechner
Tatiana S
TS
Oretskaya
Elena A
EA
Kubareva
Roland K
RK
Hartmann
D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D022661:RNA, Untranslated; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
C411065:6S RNA; D012329:RNA, Bacterial; D022661:RNA, Untranslated; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
6S RNA:pRNA hybrid stability; 6S-1 RNA; 6S-2 RNA; 6S-2 RNA release from RNAP; affinity for σA-RNAP; bsrA; bsrB; pRNA transcripts