NusG is an intrinsic transcription termination factor that stimulates motility and coordinates gene expression with NusA
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Abstract
NusA and NusG are transcription factors that stimulate RNA polymerase pausing in Bacillus subtilis . While NusA was known to function as an intrinsic termination factor in B. subtilis , the role of NusG in this process was unknown. To examine the individual and combinatorial roles that NusA and NusG play in intrinsic termination, Term-seq was conducted in wild type, NusA depletion, Δ nusG , and NusA depletion Δ nusG strains. We determined that NusG functions as an intrinsic termination factor that works alone and cooperatively with NusA to facilitate termination at 88% of the 1400 identified intrinsic terminators. Our results indicate that NusG stimulates a sequence-specific pause that assists in the completion of suboptimal terminator hairpins with weak terminal A-U and G-U base pairs at the bottom of the stem. Loss of NusA and NusG leads to global misregulation of gene expression and loss of NusG results in flagella and swimming motility defects.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on October 6 2020, follows.
Summary
The reviewers and editors were enthusiastic about the major conclusion of the study: that NusG-dependent pausing is an important factor that promotes Rho-independent transcription termination in Bacillus subtilis. Nonetheless, we felt that this conclusion can be strengthened with additional analysis and experiments that hopefully are not terribly burdensome. We believe that these additions would bring the paper to the level required for publication in eLife. The essential revisions are detailed below.
Essential Revisions
While we appreciated the careful follow-up work, we felt that the major conclusion could be strengthened by a more in-depth analysis of the genome-wide data, assuming those data …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on October 6 2020, follows.
Summary
The reviewers and editors were enthusiastic about the major conclusion of the study: that NusG-dependent pausing is an important factor that promotes Rho-independent transcription termination in Bacillus subtilis. Nonetheless, we felt that this conclusion can be strengthened with additional analysis and experiments that hopefully are not terribly burdensome. We believe that these additions would bring the paper to the level required for publication in eLife. The essential revisions are detailed below.
Essential Revisions
While we appreciated the careful follow-up work, we felt that the major conclusion could be strengthened by a more in-depth analysis of the genome-wide data, assuming those data support the role of NusG-dependent pausing in termination. Reviewers 1 and 2 give specific suggestions in their reviews. Some of these relate to the way comparisons are made between datasets, and others address specific scientific questions. Of particular relevance are analyses that test whether NusG stimulates termination at sites with (i) weak terminal base-pairs, and (ii) gaps in the U-tract. Any other analyses of the genome-wide data that support the importance of NusG-simulated pausing in termination would be valuable to include. For example, is there any evidence that NusG-dependent pause sites identified by NET-seq are associated with sites of termination?
Termination sites in vitro are consistently downstream of those observed in vivo. While it is reasonable to hypothesize that this difference is due to trimming by exonucleases, there is no experimental evidence presented to support this. To test the hypothesis, we suggest mapping termination sites by 3' RACE in RNase mutant strains for one or two of the terminators characterized in the paper. B subtilis 3' exonucleases are defined, and mutant strains have been described (e.g., Oussenko et al., 2005 J Bacteriol 187:2758; Liu et al., 2014 Mol Microbiol 94:41).
Add a figure showing the model described in the discussion (lines 332-84) for the proposed roles of NusG and NusA in intrinsic termination.
Broaden the discussion of how the study relates to prior work on intrinsic termination.
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