Summary:
Adaptation of the transcriptome to nutrient limitation and resupply
is a fundamental process in bacteria, particularly in natural
habitats. Bacterial 6S RNA, an ubiquitous and growth phasedependent
regulator of transcription, binds to RNA polymerase
(RNAP) and inhibits transcription during stationary growth.
Upon nutrient resupply, RNAP acts as an RNA-dependent RNA
polymerase by transcribing large amounts of short RNAs (pRNAs)
from 6S RNA as template, leading to dissociation of 6S RNARNAP
complexes. Whereas the majority of bacteria express a
single 6S RNA species, Bacillus subtilis encodes two 6S RNAs
(6S-1 and 6S-2) of similar secondary structure, but with different
expression profiles.
In this work, we investigated the two 6S RNAs of B. subtilis,
focusing on pRNA synthesis and its role for the function of 6S
RNA. Concurrently, we identified pRNA transcription from 6S-1
RNA in vivo using high-troughput sequencing techniques and we
developed a novel Northern hybridization protocol for detection
of pRNAs in bacterial total cellular extracts.
Our results show that the release of RNAP from 6S-1 RNA,
the functional homolog of the well investigated E. coli 6S RNA,
is regulated by stable pRNA binding. Additionally, we found
structural changes of 6S-1 RNA, induced by differences in pRNA
length in different growth phases. This specific structural change
of 6S RNA seems to be conserved among bacteria. Furthermore,
we are able to show that the two processes of RNAP release and
6S-1 RNA degradation are coupled in vivo. Taken together, our
results expand the current understanding of 6S RNA function
and provide insight into the mechanism of RNAP release from
6S RNA in bacteria.
iii
Bibliographie / References
- K M Wassarman and G Storz. 6S RNA regulates E. coli RNA polymerase activity. Cell, 101(6):613–623, Jun 2000.
- A E Trotochaud and K M Wassarman. A highly conserved 6S RNA structure is required for regulation of transcription. Nat Struct Mol Biol, 12(4):313–319, Apr 2005.
- K M Wassarman and R M Saecker. Synthesis-mediated release of a small RNA inhibitor of RNA polymerase. Science, 314(5805): 1601–1603, Dec 2006.
- F Repoila and F Darfeuille. Small regulatory non-coding RNAs in bacteria: physiology and mechanistic aspects. Biol Cell, 101 (2):117–131, Feb 2009.
- J Vogel and K Papenfort. Small non-coding RNAs and the bac- terial outer membrane. Curr Opin Microbiol, 9(6):605–611, Dec 2006.
- K M Wassarman. 6S RNA: a regulator of transcription. Mol Microbiol, 65(6):1425–1431, Sep 2007.
- J P Schlüter, J Reinkensmeier, S Daschkey, E Evguenieva- Hackenberg, S Janssen, S Jänicke, J D Becker, R Giegerich, and A Becker. A genome-wide survey of sRNAs in the sym- biotic nitrogen-fixing alpha-proteobacterium Sinorhizobium meliloti. BMC Genomics, 11:245–245, 2010. bibliography C M Sharma, S Hoffmann, F Darfeuille, J Reignier, S Findeiss, A Sittka, S Chabas, K Reiche, J Hackermüller, R Reinhardt, P F Stadler, and J Vogel. The primary transcriptome of the major human pathogen Helicobacter pylori. Nature, 464(7286): 250–255, Mar 2010.
- H D Murray, D A Schneider, and R L Gourse. Control of rRNA expression by small molecules is dynamic and nonredundant.
- C Mülhardt. Der Experimentator: Molekularbiologie/Genomics. Spek- trum Akad. Verlag, 3 edition, 2002.
- M Mandal and R R Breaker. Gene regulation by riboswitches. Nat Rev Mol Cell Biol, 5(6):451–463, Jun 2004.
- M Margulies, M Egholm, W E Altman, S Attiya, J S Bader, L A Bemben, J Berka, M S Braverman, Y J Chen, Z Chen, S B Dewell, L Du, J M Fierro, X V Gomes, B C Godwin, W He, S Helgesen, C H Ho, C H Ho, G P Irzyk, S C Jando, M L Alenquer, T P Jarvie, K B Jirage, J B Kim, J R Knight, J R Lanza, J H Leamon, S M Lefkowitz, M Lei, J Li, K L Lohman, H Lu, V B Makhijani, K E McDade, M P McKenna, E W Myers, E Nickerson, J R Nobile, R Plant, B P Puc, M T Ronan, G T Roth, G J Sarkis, J F Simons, J W Simpson, M Srinivasan, K R Tartaro, A Tomasz, K A Vogt, G A Volkmer, S H Wang, Y Wang, M P Weiner, P Yu, R F Begley, and J M Rothberg. Genome sequencing in microfabricated high-density picolitre reactors. Nature, 437 (7057):376–380, Sep 2005.
- S Suzuma, S Asari, K Bunai, K Yoshino, Y Ando, H Kakeshita, M Fujita, K Nakamura, and K Yamane. Identification and char- acterization of novel small RNAs in the aspS-yrvM intergenic region of the Bacillus subtilis genome. Microbiology, 148(Pt 8): 2591–2598, Aug 2002.
- J Livny and M K Waldor. Identification of small RNAs in diverse bacterial species. Curr Opin Microbiol, 10(2):96–101, Apr 2007.
- M G Marinus and N R Morris. Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12. J Bacteriol, 114(3): 1143–1150, Jun 1973. bibliography 177
- B Vester and J Wengel. LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry, 43 (42):13233–13241, Oct 2004.
- J Sambrook and D W Russel. Molecular Cloning. Cold Spring Harbor Laboratory Press, 3 edition, 2001.
- J M Sogo, M R Inciarte, J Corral, E Viñuela, and M Salas. Rna polymerase binding sites and transcription map of the DNA of bacillus subtilis phage phi29. J Mol Biol, 127(4):411–436, Feb 1979.
- S G Landt, E Abeliuk, P T McGrath, J A Lesley, H H McAdams, and L Shapiro. Small non-coding RNAs in Caulobacter cres- centus. Mol Microbiol, 68(3):600–614, May 2008.
- K M Wassarman, A Zhang, and G Storz. Small RNAs in Es- cherichia coli. Trends Microbiol, 7(1):37–45, Jan 1999.
- J F Milligan and O C Uhlenbeck. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol, 180:51–62, 1989.
- J Vogel and E G Wagner. Target identification of small noncoding RNAs in bacteria. Curr Opin Microbiol, 10(3):262–270, Jun 2007.
- J M Lopez, C L Marks, and E Freese. The decrease of guanine nucleotides initiates sporulation of Bacillus subtilis. Biochim Biophys Acta, 587(2):238–252, Oct 1979.
- L Krásny, H Tiserová, J Jonák, D Rejman, and H Sanderová. The identity of the transcription +1 position is crucial for changes in gene expression in response to amino acid starvation in Bacillus subtilis. Mol Microbiol, 69(1):42–54, Jul 2008.
- J Livny, H Teonadi, M Livny, and M K Waldor. High-throughput, kingdom-wide prediction and annotation of bacterial non- coding RNAs. PLoS One, 3(9), 2008.
- K B Mullis, F Ferre, and R A Gibbs. The polymerase chain reaction. Birkäuser Boston, 1994.
- N R Mattatall and K E Sanderson. Salmonella typhimurium LT2 possesses three distinct 23S rRNA intervening sequences. J Bacteriol, 178(8):2272–2278, Apr 1996.
- C A Lee, M J Fournier, and J Beckwith. Escherichia coli 6S RNA is not essential for growth or protein secretion. J Bacteriol, 161 (3):1156–1161, Mar 1985.
- A Sittka, S Lucchini, K Papenfort, C M Sharma, K Rolle, T T Binnewies, J C Hinton, and J Vogel. Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq. PLoS Genet, 4(8), 2008.
- J W Nicol, G A Helt, S G Blanchard, A Raja, and A E Loraine. The Integrated Genome Browser: free software for distribution and exploration of genome-scale datasets. Bioinformatics, 25 (20):2730–2731, Oct 2009.
- T Mizuno, M Y Chou, and M Inouye. A unique mechanism regulating gene expression: translational inhibition by a com- plementary RNA transcript (micRNA). Proc Natl Acad Sci U S A, 81(7):1966–1970, Apr 1984.
- L Krásny and R L Gourse. An alternative strategy for bacte- rial ribosome synthesis: Bacillus subtilis rRNA transcription regulation. EMBO J, 23(22):4473–4483, Nov 2004.
- R P Novick and E Geisinger. Quorum sensing in staphylococci. Annu Rev Genet, 42:541–564, 2008.
