6S RNA Regulates E. coli RNA Polymerase Activity (original) (raw)
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6S RNA is a widespread regulator of eubacterial RNA polymerase that resembles an open promoter
2005
6S RNA is an abundant noncoding RNA in Escherichia coli that binds to 70 RNA polymerase holoenzyme to globally regulate gene expression in response to the shift from exponential growth to stationary phase. We have computationally identified >100 new 6S RNA homologs in diverse eubacterial lineages. Two abundant Bacillus subtilis RNAs of unknown function (BsrA and BsrB) and cyanobacterial 6Sa RNAs are now recognized as 6S homologs. Structural probing of E. coli 6S RNA and a B. subtilis homolog supports a common secondary structure derived from comparative sequence analysis. The conserved features of 6S RNA suggest that it binds RNA polymerase by mimicking the structure of DNA template in an open promoter complex. Interestingly, the two B. subtilis 6S RNAs are discoordinately expressed during growth, and many proteobacterial 6S RNAs could be cotranscribed with downstream homologs of the E. coli ygfA gene encoding a putative methenyltetrahydrofolate synthetase. The prevalence and robust expression of 6S RNAs emphasize their critical role in bacterial adaptation. 6604.
RNA 6S. Efectos sobre la regulación de la expresión génica en E. coli.
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6S RNA Function Enhances Long-Term Cell Survival
Journal of Bacteriology, 2004
6S RNA was identified in Escherichia coli >30 years ago, but the physiological role of this RNA has remained elusive. Here, we demonstrate that 6S RNA-deficient cells are at a disadvantage for survival in stationary phase, a time when 6S RNA regulates transcription. Growth defects were most apparent as a decrease in the competitive fitness of cells lacking 6S RNA. To decipher the molecular mechanisms underlying the growth defects, we have expanded studies of 6S RNA effects on transcription. 6S RNA inhibition of 70 -dependent transcription was not ubiquitous, in spite of the fact that the vast majority of 70 -RNA polymerase is bound by 6S RNA during stationary phase. The 70 -dependent promoters inhibited by 6S RNA contain an extended ؊10 promoter element, suggesting that this feature may define a class of 6S RNA-regulated genes. We also discovered a secondary effect of 6S RNA in the activation of S -dependent transcription at several promoters. We conclude that 6S RNA regulation of both 70 and S activities contributes to increased cell persistence during nutrient deprivation.
Nucleic Acids Research, 2011
6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Ep 70 in Escherichia coli, Ep A in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Ep 70 during exit from stationary phase by a process in which 6S RNA serves as a template for Ep 70 to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Ep A prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNAsynthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.
Binding and release of the 6S transcriptional control RNA
RNA, 2010
6S RNA is an important noncoding RNA that regulates eubacterial transcription. In Escherichia coli this RNA binds to the s 70 RNA polymerase holoenzyme and is released by the synthesis of a short product RNA. In order to determine how binding and release are controlled by the 6S RNA sequence, we used in vitro selection to screen a high diversity library containing ;4 3 10 12 sequences for functional 6S RNA variants. Residues critical for binding were found to be located in a ''-35'' region upstream of the 6S RNA transcription bubble mimic structure. Mutating these phylogenetically conserved residues invariably led to decreases in binding and removing them abolished binding, implicating these nucleotides in a biologically important interaction with the Es 70 complex. Interestingly, mutation of phylogenetically conserved ''-10'' residues that were also upstream of the site of pRNA synthesis was found to influence 6S RNA release rates in addition to modulating -35 binding. These results indicate how 6S RNA -35 binding to s 70 RNA polymerase holoenzyme can regulate expression from ''strong'' and ''weak'' -35 DNA promoters and suggest that 6S RNA release rates have been fine tuned over evolutionary time so as to correctly regulate cellular levels of transcription. .
Journal of Bacteriology, 1991
Igarashi et al. (K. Igarashi, N. Fujita, and A. Ishihama, Nucleic Acids Res. 17:8755-8765, 1989) reported that the omega (omega) subunit of Escherichia coli RNA polymerase was required for stringent control as judged by in vitro transcription assays in the presence and absence of guanosine 3',5'-bispyrophosphate (ppGpp). This conclusion predicts that a deletion of the omega gene (designated rpoZ or spoS) should show a relaxed RNA control phenotype in vivo. However, we find that wild-type stringent control of stable RNA accumulation is unaffected by a spoS null allele that abolishes cellular production of the omega protein. We conclude that omega protein is not necessary for the operation of the stringent RNA control response.
Escherichia coli 6S RNA gene is part of a dual-function transcription unit
Journal of Bacteriology, 1985
The gene coding for the metabolically stable 6S RNA of Escherichia coli has been cloned, sequenced, and partially characterized in expression analyses. The DNA sequence results confirm the accuracy of the previously established RNA sequence and, with genomic hybridization data, reveal that there is only one copy of the 6S DNA in the chromosome. Consistent with its relaxed mode of expression, the promoter region of the 6S RNA gene was found to lack the hypothetical GC-rich discriminator domain common to other stable RNA genes under stringent control. The sequence results also reyealed the occurrence of a 540-base-pair open reading frame immediately downstream from the 6S RNA coding region. Results from the expression analyses show that the protein and RNA coding regions are cotranscribed in vitro and that the open reading frame is translated in vivo.
Journal of Bacteriology, 2000
Adaptation of bacterial cells to diverse habitats relies on the ability of RNA polymerase to respond to various regulatory signals. Some of these signals are conserved throughout evolution, whereas others are species specific. In this study we present a comprehensive comparative analysis of RNA polymerases from two distantly related bacterial species, Escherichia coli and Bacillus subtilis, using a panel of in vitro transcription assays. We found substantial species-specific differences in the ability of these enzymes to escape from the promoter and to recognize certain types of elongation signals. Both enzymes responded similarly to other pause and termination signals and to the general E. coli elongation factors NusA and GreA. We also demonstrate that, although promoter recognition depends largely on the subunit, promoter discrimination exhibited in species-specific fashion by both RNA polymerases resides in the core enzyme. We hypothesize that differences in signal recognition are due to the changes in contacts made between the  and  subunits and the downstream DNA duplex. on June 23, 2015 by guest http://jb.asm.org/ Downloaded from on June 23, 2015 by guest http://jb.asm.org/ Downloaded from on June 23, 2015 by guest http://jb.asm.org/ Downloaded from
Nucleic Acids Research, 2011
Specific promoter recognition by bacterial RNA polymerase is mediated by p subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, p 70 (the housekeeping p subunit) and p S (an alternative p subunit mostly active during slow growth) recognize almost identical promoter sequences, thus raising the question of how promoter selectivity is achieved in the bacterial cell. To identify novel sequence determinants for selective promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA polymerase saturated either with p 70 (Ep 70) or with p S (Ep S) using the whole Escherichia coli genome as DNA template. We found that Ep 70 , in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, Ep S efficiently transcribes genes involved in stress responses, secondary metabolism as well as RNAs from intergenic regions with yet-unknown function. Promoter sequence comparison suggests that, in addition to different conservation of the À35 sequence and of the UP element, selective promoter recognition by either form of RNA polymerase can be affected by the A/ T content in the À10/+1 region. Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the À10/+1 region could improve promoter recognition by Ep S .