Funktion und Metabolismus der regulatorischen, nicht-kodierenden 6S RNAs aus Bacillus subtilis
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Philipps-Universität Marburg
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Abstract
Bacterial 6S RNAs are short non-coding RNAs that regulate transcription by directly binding to
the active site of the primary RNA polymerase holoenzyme (σA-RNAP). Their highly conserved
secondary structure, consisting of two irregular helical arms interrupted by regions of unpaired
nucleotides that flank a large, mostly single-stranded region termed central bulge, is crucial for
the interaction with RNAP. Thereby, 6S RNAs mimic an open DNA promotor structure,
allowing them to compete with DNA promotors for binding to the active site of RNAP and to
inhibit transcription of cellular genes. Furthermore, 6S RNAs can also serve as transcription
templates for RNAP. In this RNA-dependent RNA polymerization, short product RNAs
(pRNAs) are synthesized that, if long enough, remain stably bound to the 6S RNA template
and induce refolding of 6S RNA. As a consequence, the 6S RNA::pRNA complex dissociates
from RNAP and is degraded, while RNAP can resume transcription of housekeeping genes.
A special feature of the Gram-positive model organism Bacillus subtilis studied in this work is
the expression of two 6S RNA paralogues, 6S-1 and 6S-2 RNA. While 6S-1 RNA as the
canonical 6S RNA reaches its maximum concentration during stationary growth, 6S-2 RNA
levels peak during exponential growth. In comparison to corresponding laboratory strains,
6S RNA deletions in the undomesticated B. subtilis wild-type strain NCIB 3610 exhibited more
distinct phenotypes. While deletion of the 6S-1 RNA gene (ΔbsrA) showed reduced growth in
late stationary phase as it was also observed in laboratory strains, a prolonged lag phase was
ascertained under osmotic, oxidative and alkaline stress for the 6S-1&6S-2 RNA double
deletion strain (ΔbsrA/bsrB) and in contrast to laboratory strains. Effects of the 6S-2 RNA
deletion (ΔbsrB) were most pronounced, resulting in significantly increased biofilm formation,
reduced swarming activity and accelerated spore formation.
In this work, potential binding of 6S RNAs to alternative RNAP holoenzymes assembled with
σB, σD or σF was tested, revealing a specific 6S-1 RNA interaction with σB- and σF-RNAP
holoenzymes. No interaction was observed with σD-RNAP. Compared with 6S-1 RNA binding
to the primary σA-RNAP, affinity to the aforementioned alternative holoenzymes was reduced,
however, it was shown for the first time that 6S RNA binding in B. subtilis is not restricted to
the primary σA-RNAP. In contrast, no binding to alternative RNAP holoenzymes was observed
for 6S-2 RNA under the tested conditions. In addition, 6S-2 RNA showed enhanced inhibitory
activity on transcription at weaker σA-dependent promoters (veg core promoter), suggesting
that 6S-2 RNA is more likely to be effective at weaker promoters with lower affinity for σARNAP
in vivo. Furthermore, the influence of different amino acids and structural elements of
the primary sigma factor σA on 6S RNA binding was investigated. For this purpose, different
σA mutants were tested in in vitro transcription assays as well as in electrophoretic mobility
shift assays (EMSAs). These experiments revealed striking differences between the RNAP
holoenzymes from E. coli and B. subtilis. Unexpectedly, successive mutation of basic amino
acids in the σ4.2 region as well as deletion of the entire σ4 domain (σA Δ4.1+4.2+C) surprisingly
did not show a reduced 6S RNA-mediated transcription inhibition with the B. subtilis holo-
RNAP. In contrast, no promoter DNA binding and thus no transcription product could be
detected with a chimeric RNAP holoenzyme consisting of E. coli core RNAP and B. subtilis σA
mutant. Domain σ4, which is essential for both promoter DNA and 6S RNA binding in E. coli,
therefore appears to be less important in the B. subtilis system.
In an additional part project, processing and decay of B. subtilis 6S RNAs were investigated.
For this purpose, Northern blot analyses and RNA sequencing were carried out with different
RNase knockout strains. These showed cleavage of the 5’-precursor of pre-6S-1 RNA by
RNase J1 as well as processing of its 3’-end by RNase PH. Maturation of 6S-1 RNA is not
essential for 6S-1 pRNA synthesis, but rather regulates 6S-1 RNA degradation. Furthermore,
endonucleolytical cleavage in the apical loop region of 6S-1 RNA leads to an accumulation of
5'- and 3'-fragments of approximately equal length in stationary phase, which are further
degraded by 3'-exoribonucleases during outgrowth. In contrast, degradation of 6S-2 RNA is
initiated by cleavage in the 5'-central bulge and subsequent degradation of the 3'-fragment by
the 5'-3'-exonucleolytic activity of RNase J1. As with 6S-1 RNA, the 6S-2 RNA 3’-end was
trimmed by RNase PH. A specific function of RNase Y could not be demonstrated for either of
the 6S RNAs, but involvement of the enzyme in the degradation of both RNAs in wild-type cells
cannot be excluded because substrate specificities of RNases Y, J1 and J2 overlap. Moreover,
the four 3'-exonucleases (RNase R, PNPase, YhaM, RNase PH) of B. subtilis, especially
RNase PH, are responsible for the degradation of 6S-1 pRNAs.
Furthermore, protocols for the preparation of His-tagged 6S RNA-free B. subtilis RNA
polymerase and His-tagged B. subtilis σA as well as for Northern blot detection of tiny RNAs
(8-15 nt) were established and published.
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Dates
Created: 2023Issued: 2024-09-16Updated: 2024-09-16
Faculty
Fachbereich Pharmazie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
interaction analysisRNA processingRNA decaysigma factors
DFG-subjects
InteraktionsanalyseBacillus subtilis6S RNAsRNase J1RNA-ProzessierungSigma-FaktorenRNARNA-AbbauRNA-Polymerase
DDC-Numbers
615
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Wiegard, Jana Christin: Funktion und Metabolismus der regulatorischen, nicht-kodierenden 6S RNAs aus Bacillus subtilis. : Philipps-Universität Marburg 2024-09-16. DOI: https://doi.org/10.17192/z2023.0487.
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This item has been published with the following license: In Copyright