Annotation and evolutionary relationships of a small regulatory RNA gene micF and its target ompF in Yersinia species

Abstract

Background: micF RNA, a small regulatory RNA found in bacteria, post-transcriptionally regulates expression of outer membrane protein F (OmpF) by interaction with the ompF mRNA 5'UTR. Phylogenetic data can be useful for RNA/RNA duplex structure analyses and aid in elucidation of mechanism of regulation. However micF and associated genes, ompF and ompC are difficult to annotate because of either similarities or divergences in nucleotide sequence. We report by using sequences that represent "gene signatures" as probes, e.g., mRNA 5'UTR sequences, closely related genes can be accurately located in genomic sequences.

Results: Alignment and search methods using NCBI BLAST programs have been used to identify micF, ompF and ompC in Yersinia pestis and Yersinia enterocolitica. By alignment with DNA sequences from other bacterial species, 5' start sites of genes and upstream transcriptional regulatory sites in promoter regions were predicted. Annotated genes from Yersinia species provide phylogenetic information on the micF regulatory system. High sequence conservation in binding sites of transcriptional regulatory factors are found in the promoter region upstream of micF and conservation in blocks of sequences as well as marked sequence variation is seen in segments of the micF RNA gene. Unexpected large differences in rates of evolution were found between the interacting RNA transcripts, micF RNA and the 5' UTR of the ompF mRNA. micF RNA/ompF mRNA 5' UTR duplex structures were modeled by the mfold program. Functional domains such as RNA/RNA interacting sites appear to display a minimum of evolutionary drift in sequence with the exception of a significant change in Y. enterocolitica micF RNA.

Conclusions: Newly annotated Yersinia micF and ompF genes and the resultant RNA/RNA duplex structures add strong phylogenetic support for a generalized duplex model. The alignment and search approach using 5' UTR signatures may be a model to help define other genes and their start sites when annotated genes are available in well-defined reference organisms.

Figure 1

Alignment of ompF mRNA 5' UTRs from 6 bacterial species. Putative 5' end nucleotides (position one) of Yersinia species ompF transcripts were determined by sequence alignment. Sequences were aligned by the ClustalV method using the DNASTAR Inc MegAlign program. OpnP is the ompF homolog in X. nematophilus. Color bar indicates degree of similarity at each position with red signifying 100% identity, orange signifying that 5 out of 6 nucleotides are the same, green shows partial similarity whereas purples depict poor identity. The consensus sequence is shown under the color bar.

Figure 2

The % identity between bacterial ompF mRNA 5' UTR sequences shown in Figure 1.

Figure 3

Alignment of ompC 5' UTR sequences (by J. Hein method). Putative 5' start sites of Yersinia species ompC were also determined by sequence alignment. The consensus sequence is shown below the color bar.

Figure 4

Percent identity between ompC 5' UTR sequences from five bacterial species.

Figure 5

Alignment of micF gene sequences (by J. Hein method). The consensus sequence is shown below the color bar.

Figure 6

Percent sequence identity in micF bacterial genes.

Figure 7

Phylogenetic tree of ompF mRNA 5' UTR sequences determined by DNASTAR program.

Figure 8

Phylogenetic tree of micF sequences determined by DNASTAR program.

Figure 9

Alignment of sequences upstream of ompC and micF (promoter and transcription regulatory region). J. Hein alignment method was used, however positions 197–213 and 246–261 were aligned by eye to reflect known homologies. The consensus (Majority) sequence is shown above alignments. Promoters (P-10, P-35) and transcription factor binding sites are shown in color. ompC and micF are transcribed in opposite directions (shown with arrows).

Figure 10

Y. pestis ompF mRNA 5' UTR/micF RNA duplex model. Secondary structures determined by mfold program of Zuker and co-workers. D. Stewart and M. Zuker graphics program was used (web site: ). The Y. pestis duplex structure represents the first alternate structure by mfold modeling. Numbers 1–3 in the figure refer to inter-molecular stems formed by ompF mRNA 5' UTR and micF RNA sequences. Letters a-c refer to intra-molecular micF or ompF mRNA 5'UTR stem loops.

Figure 11

Y. enterocolitica ompF mRNA 5' UTR/micF RNA duplex model. The Y. enterocolitica duplex is structure one by mfold modeling. Numbers 1–3 in the figure refer to inter-molecular stems formed by ompF mRNA 5' UTR and micF RNA sequences. Letters a-c refer to intra-molecular micF or ompF mRNA 5'UTR stem loops. Positions shown in blue color are those that differ between Y. enterocolitica and Y. pestis micF RNAs.

Figure 12

Diagrammatic representation of duplex models from four bacterial species. The E. coli RNA/RNA duplex model was determined by structure probing [2]. The S. marcescens RNA/RNA duplex model is according to the long range pairing algorithm of [38,8].