Regulation of gene expression by small RNAs

Small non-coding RNAs regulate gene expression at the post-transcriptional level by binding to target mRNAs and inhibiting translation and/or inducing degradation of the message. Regulatory RNAs were first discovered in prokaryotes but are widely found in both prokaryotes and eukaryotes. In E. coli approximately 60 non-coding RNA genes have been detected. Most RNAs are encoded in intergenic regions of the genome and expression of the RNA gene is induced by environmental signals. Mechanisms of post- transcriptional regulation by small RNAs, their abundance in various bacteria, and their evolutionary origins, as well as the mechanism of transcriptional activation of regulatory RNA genes, pose challenging problems.

For the past 20 years we have largely concentrated on investigating the genetics of the micF gene, the structural properties of the micF RNA transcript and its functional role. micF was first discovered by Masayori Inouye and Takeshi Mizuno in the early 1980s.

Current work involves use of bioinformatics analysis to find regulatory RNA genes in bacteria with specialized habitats. Obligate endosymbionts such Buchnera and Wiggleswothia which live in a protected environment may have few or no regulatory RNA genes Regulatory RNA genes may be more prevalent in bacteria that are free living where survival is dependent on the ability to make rapid adjustments in response to environmental stress.

Annotation of genes

The rapid elucidation of microbial genomic sequences poses a challenge in gene annotation and assignment of transcriptional start sites. Without experimental data, incorrect annotations can be made as well as erroneous determination of gene start sites. This is especially true for genes that are evolutionarily and structurally related such as the bacterial porin genes, ompF and ompC . However, when gene promoter sites, transcript 5'UTR sequences, or signatures within genes from reference organisms are used, an accurate assignment of a gene as well as a prediction of its start site can be made by a comparative approach. These regions serve specific functions in molecular processes, e.g., several 5' UTRs of mRNA transcripts are mRNA stability determinants. Thus they can display sequence and/or secondary structure signatures and these defined segments can be more useful than using entire gene sequences for annotations. We have successfully used 5' UTR sequences to annotate the evolutionary related porin genes in Yersinia species and Photorhabdus luminescens. In the future, a combination of sequence and secondary structure motifs will be utilized is for additional searches and annotations, e.g., annotation of regulatory RNA genes.

Repetitive sequences in intergenic regions in bacteria

The bacterial genome displays numerous examples of multiple duplications of nucleotide sequences in intergenic regions. While searching for the non-coding RNA gene itsR in Yersinia species genomes in a locus analogous to the locus that itsR is found in E coli, we detected a 170 nucleotide insert in this region. This sequence is a fragment (68%) of a protein coding gene YPTB2112 present in Yersinia pseudotuberculosis. Fragments of YPTB 2112 are found in multiple intergenic regions of the chromosome (~60 duplications). These sequences are highly conserved in several Yersinia sp. The putative YPTB2112 protein is of unknown function.

What advantage are repetitive DNA sequences to the organism? We are using a bioinformatics approach to ascertain functional roles and/or possible evolutionary advantages of the 170 nt repeat sequence in Yersinia as well as other repetitive sequences in bacterial genomes.