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RybB

RybB is a small regulatory RNA containing 81 nucleotides in length. The expression of this sRNA is related with the cell envelope stress and dependent on an alternative sigma factor sE. The cellular envelope is an important line of defence for the bacterial cell and the monitoring of its integrity is essential for its fight against antibiotics. This makes RybB an important small regulatory RNA when it comes to bacterial resistance against antibiotics. During cell envelope stress, sigma factor sE is activated and stimulates the expression of RybB, which increases in stationary phase. This regulatory mechanism has an autoregulatory loop: RybB indirectly represses the synthesis of sE.

The interaction of specific proteins will also regulate the transcription of the rybB gene. DksA, during the stationary phase, activates the binding of RNA polymerase to the rybB promoter region, stimulating rybB transcription. This activation is only possible with the presence of ppGpp. NsrR is a transcription repressor, which prevents the initiation of the transcription of the rybB gene.

RybB sRNA has various regulatory targets, which were predicted by comparative genomics, analysing the accessible regions in both the sRNA and its target mRNA.

Hfq is a protein that forms complexes with small RNAs, which have major roles in the control of bacterial genetic expression. RybB is one of the sRNAs that interact with this protein, in order to target specific mRNA sequences. This interaction has been probed using Salmonella components, which is a genetic evidence that the conserved 5´end of RybB acts as an autonomous multiple-target binding domain for many omp RNAs by short (≥7 bp) Watson-Crick pairing.

The overexpression of rybB has several effects in the cell. Firstly, it causes increased expression of rpoS in minimal media and decreases levels of outer membrane proteins (like ompC and ompW), rpoE, and rybB itself. Secondly, rescue of the growth and viability phenotype of sE depletion, and leads to cell lysis in stationary phase. Finally, the overexpression decreases biofilm formation and expression of type I fimbriae. Therefore, a rybB deletion mutant is less sensitive to cell envelope stress than a wild type cell (Hobbs10), and as a result, appears unable to establish long-term stationary phase.

Usage and Biology

This synthetic version of RybB follows the same principles as its wild type partner to down-regulate gene expression. This modified construct, however, can target any gene of interest due to its sequence which contains a BaeI restriction enzyme site. After digestion with this restriction enzyme and ligation with a target sequence, RybB can down-regulate the expression of the gene that contains the complementary regions for its targeting sequence. Ligation with the desired targeting sequence does not create any scar in the sRNA sequence, therefore allowing its use for a broad range of gene targets as well as with any biobricks that may benefit from its functions.

Sequence and Features