MicF: an antisense RNA gene involved in response of Escherichia coli to global stress factors.

The micF gene is a stress response gene found in Escherichia coli and related bacteria that post-transcriptionally controls expression of the outer membrane porin gene ompF. The micF gene encodes a non-translated 93 nt antisense RNA that binds its target ompF mRNA and regulates ompF expression by inhibiting translation and inducing degradation of the message. In addition, other factors, such as the RNA chaperone protein StpA also play a role in this regulatory system. Expression of micF is controlled by both environmental and internal stress factors. Four transcriptional regulators are known to bind the micF promoter region and activate micF expression. The crystal structure of one these transcriptional activators, Rob, complexed with the micF promoter has been reported. Here, we review new developments in the micF regulatory network.

Targeting the expression of anti-apoptotic proteins by antisense oligonucleotides.

Antisense oligonucleotide (ASO) biotechnology has been widely used to inhibit the expression of proteins involved in human disease. ASOs are designed to bind messenger RNA transcripts via Watson-Crick base-pairing and inhibit synthesis of targeted proteins. These proteins include protein kinases, growth factors, glutamate receptors, anti-apoptotic proteins, and proteins involved in genetic disorders. Non-mRNA targets such as the RNA component of the telomerase enzyme are also being explored. Pre-clinical and clinical trials using ASO biotechnology have progressed with standard ASOs such as phosphorothioates, but newer ASO analogs are rapidly being developed with the idea of enhancing specificity and biological activity. A current major research thrust is the design and testing of antisense oligonucleotides as anti-cancer drugs. The primary focus of this review is an analysis of recent uses of ASO biotechnology to inhibit anti-apoptotic gene expression in tumor cells.

Cleavage of mitochondria-like transfer RNAs expressed in Escherichia coli.

Mitochondrial (mt) transfer RNAs (tRNAs) often harbor unusual structural features causing their secondary structure to differ from the conventional cloverleaf. tRNAs designed with such irregularities, termed mt-like tRNAs, are active in Escherichia coli as suppressors of reporter genes, although they display low steady-state levels. Characterization of fragments produced during mt-like tRNA processing in vitro and in vivo suggests that these RNAs are not fully processed at their 5' ends and are cleaved internally. These abnormal processing events may account for the low levels of mature mt-like RNAs in vivo and are most likely related to defective processing by RNase P.

Natural antisense RNA/target RNA interactions: possible models for antisense oligonucleotide drug design.

Current antisense oligonucleotides designed for drug therapy rely on Watson-Crick base pairing for the specificity of interactions between antisense and target molecules. However, thermodynamically stable duplexes containing non-Watson-Crick pairs have been formed with synthetic oligonucleotides. There are also numerous examples of non-canonical base pairs that participate in stable intra- and inter-molecular RNA/RNA pairing in prokaryotic and eukaryotic cells. Several natural antisense RNA/target RNA duplexes contain looped-out and bulged positions as well as non-canonical pairs as exemplified by formation of the Escherichia coli antisense micF RNA/ompF mRNA duplex. Secondary structures and the phylogenetic conservation of nucleotide sequences are well characterized in this system. Natural antisense/ target interactions may serve as models for determining possible and optimal antisense/target interactions in oligonucleotide drug design.

Antisense micF RNA and 5'-UTR of the target ompF RNA: phylogenetic conservation of primary and secondary structures.

Outer membrane protein F (OmpF) found in E. coli and related bacteria is post-transcriptionally regulated by antisense micF RNA. During down regulation of ompF expression, micF RNA binds to the 5' UTR of ompF mRNA, blocks translation of the message, and also participates in the chemical destabilization of the ompF mRNA. Only about one third of the micF RNA sequence binds the target ompF mRNA. Phylogenetic analyses of micF RNA and ompF mRNA show: 1) a high degree of conservation of nucleotide sequence in regions of both RNAs involved in RNA/RNA interaction, 2) a low nucleotide sequence conservation but high degree of secondary structure conservation in regions of antisense and target RNAs not involved in the RNA/RNA interaction. Whereas conserved sequences are associated with RNA/RNA binding and blockage of translation, conservation of secondary structure may be related to protein interactions associated with chemical destabilization of the message.

micF RNA is a substrate for RNase E.

Ribonuclease E (RNase E) is known to play an important role in mRNA decay and RNA processing in Escherichia coli. While several substrates for RNase E have been identified, the specificity for the recognition and cleavage sites has not been completely determined. In this study, micF RNA, an antisense RNA found in E. coli and related bacteria, was found to be a substrate for RNase E in vitro. Two cleavage sites were mapped, and both are found in the sequence context UA/UUU and are located within 10 nucleotides upstream of stem-loop structures. These results help define a generalized RNase E cleavage/recognition pattern.

High sensitivity of Mycobacterium species to the bactericidal activity by polylysine.

Bactericidal effects of polylysine on different bacterial species were measured. Marked differences in sensitivity were observed. Based on the concentration of polylysine required to reduce cell viability by 50%, Mycobacterium smegmatis and Mycobacterium tuberculosis were found to be the most sensitive and Escherichia coli the most resistant. In addition, two Gram-positive organisms, Staphylococcus epidermidis and Streptococcus salivarius exhibited significant differences in sensitivity which suggests that the relationship between sensitivity towards polylysine and bacterial cell type is not necessarily a function of the overall cell envelope structure. The high sensitivity of mycobacteria suggests the possible use of polylysine, or a conjugate of polylysine and another agent in anti-mycobacterial drug design.

Secondary structures of Escherichia coli antisense micF RNA, the 5'-end of the target ompF mRNA, and the RNA/RNA duplex.

The Escherichia coli micF RNA is a prototype for a class of antisense RNAs encoded by genes at different loci from those that code for their target RNAs. RNAs in this class exhibit only partial complementarity to their targets. micF RNA binds to and regulates the stability of ompF mRNA in response to a variety of environmental stimuli. The secondary structures of micF RNA, ompF-213 mRNA (a segment containing the 213 nucleotides at the 5'-terminus of the target message), and the micF RNA/ompF-213 mRNA duplex were analyzed in vitro by partial digestion with structure-specific ribonucleases and chemical modification. Both micF RNA and ompF mRNA have single-stranded 5'-ends and contain stable stem-loop structures. Strong phylogenetic support for the proposed secondary structure for E. coli micF RNA is provided by a comparison of structural models derived from micF sequences from related bacteria. The micF RNA/ompF-213 mRNA duplex interaction appears to involve only a short segment of micF RNA. Unfolding of only one stem-loop of micF RNA and a minor stem-loop of ompF-213 mRNA appears to be necessary to form the duplex. The probing data suggest that the Shine-Dalgarno sequence and AUG start codon of ompF mRNA, found in single-stranded regions in the free message, are base-paired to micF RNA in the RNA/RNA duplex.

Regulation of gene expression by trans-encoded antisense RNAs.

Members of a class of antisense RNAs are encoded by genes that are located at loci other than those of their target genes. Three examples of antisense RNA genes are discussed here. micF is found in Escherichia coli and other bacteria and functions to control outer membrane protein F levels in response to environmental stimuli. dicF is also found in E. coli and is involved in the regulation of cell division. lin-4 is found in the nematode Caenorhabditis elegans and functions during larval development. Nucleotide sequences of at least two of these genes appear to be phylogenetically conserved. The trans-encoded antisense RNAs are small RNAs which display only partial complementarity to their target RNAs. Models for RNA/RNA interactions have been proposed. It is possible that currently known unlinked antisense RNA genes are part of a larger class of heretofore undiscovered regulatory RNA genes. Possible ways of detecting other unlinked antisense RNA genes are discussed.

The regulatory RNA gene micF is present in several species of gram-negative bacteria and is phylogenetically conserved.

micF RNA post-transcriptionally regulates Escherichia coli outer membrane protein F (OmpF), in response to temperature increase and other environmental stress conditions, by binding to ompF mRNA and destabilizing the message. Southern analyses show that the micF gene is present in related Gram-negative bacteria, including Salmonella typhimurium, Klebsiella pneumoniae, and Pseudomonas aeruginosa. In addition, Northern analyses indicate that micF RNA and ompF mRNA levels are thermally regulated in several related species in a manner similar to the thermoregulation in Escherichia coli. DNA sequences from Salmonella typhi, Salmonella typhimurium, and Klebsiella pneumoniae show greater than 96% homology in the micF gene when compared to the Escherichia coli micF sequence. Upstream of micF, sequences show considerable variation, although several distinct regions are highly conserved. Some of these conserved regions correspond to known binding sites for the transcription factor OmpR and the DNA-binding protein integration host factor. In addition, E. coli micF RNA incubated with protein extracts from other species forms heterologous ribonucleoproteins (RNPs). The formation of these heterologous RNPs indicates both the presence of micF RNA-binding protein homologues in other species and a conservation of RNA-protein recognition sites. This work demonstrates that the micF RNA regulatory system is present in other Gram-negative bacterial species and that this system appears to be phylogenetically conserved.

micF RNA binds to the 5' end of ompF mRNA and to a protein from Escherichia coli.

micF RNA regulates the levels of outer membrane protein F (OmpF) in Escherichia coli in response to temperature increase and other stress conditions by decreasing the levels of ompF mRNA (Andersen et al., 1989). A 93-nucleotide micF RNA was synthesized in vitro directly from polymerase chain reaction generated DNA which was designed to contain a functional T7 RNA polymerase promoter upstream of the micF RNA gene and an appropriate restriction site for transcription termination. A transcript (150 nucleotides) containing the ribosomal binding domain of ompF mRNA messenger was synthesized in vitro from the ompF gene cloned into a T7 expression vector. A stable duplex was formed between micF RNA and the 150-nucleotide 5' transcript of ompF mRNA after incubation at 37 degrees C in a physiological buffer. The melting curve of the duplex formed by micF RNA and 150-nucleotide transcript revealed a Tm of 56 degrees C and a delta Tm that spans about 20 degrees C; both are consistent with the proposed structure for the micF/ompF duplex. In addition, as determined by competition studies and UV cross-linking/label-transfer analyses, an E. coli protein was found to bind specifically to micF RNA. The protein also bound weakly to the 150-nucleotide ompF transcript. The data are the first to demonstrate the complex between micF RNA and the 5' end of ompF mRNA and suggest that in vivo a micF ribonucleoprotein (RNP) particle may participate in the destabilization ompF mRNA during thermoregulation of OmpF porin.

micF RNA in ompB mutants of Escherichia coli: different pathways regulate micF RNA levels in response to osmolarity and temperature change.

The repressor RNA, micF RNA, is regulated by temperature, osmolarity, and other stress conditions during growth of Escherichia coli. Northern (RNA) blot analyses showed that levels of micF RNA differ widely in various ompB mutant strains when cells are grown at 24 degrees C in LB broth. For example, relative to the parental strain MC4100, the ompR101 mutant strain (which contains no functional OmpR) had about a 10-fold reduction in micF RNA, whereas the envZ11 strain showed about a 5-fold increase. At 37 degrees C, however, micF RNA levels in the ompR101 and envZ11 strains and other ompB mutants differed by less than two-fold compared with the level in strain MC4100, thus indicating that a factor(s) independent of the ompB locus regulates micF RNA expression with temperature increase and that there is an additional control mechanism(s) which maintains the levels of micF RNA in these mutants close to that of the wild type during growth at high temperatures. In a plasmid strain containing the micF gene but without the upstream OmpR-binding site, steady-state levels of micF RNA increased with temperature increase but did not change with osmolarity increase. This showed that osmolal regulation but not temperature regulation of micF depends on these upstream sequences and suggested that while osmolal regulation of the micF gene depends on OmpR, thermal regulation does not.

The function of micF RNA. micF RNA is a major factor in the thermal regulation of OmpF protein in Escherichia coli.

The role of chromosomally derived micF RNA as a repressor of outer membrane protein OmpF of Escherichia coli was examined for various growth conditions. Levels of micF RNA as determined by Northern analyses are found to increase in response to cell growth at high temperature, in high osmolarity or in the presence of ethanol. After a switch to higher growth temperature, the levels of ompF mRNA and of newly synthesized OmpF decrease with time in E. coli strain, MC4100 but these decreases are not observed in isogenic micF deletion strain, SM3001. In addition, while levels of ompF mRNA are substantially reduced in both strains in response to high osmolarity or ethanol at 24 degrees C, the reduced levels in the parental strain are still 4-5-fold lower compared with the micF deletion strain. These findings indicate that chromosomally derived micF RNA plays a major role in the thermal regulation of OmpF and represses OmpF synthesis in response to several environmental signals by decreasing the levels of ompF mRNA. Analyses of the effect of a multicopy micF plasmid on the levels of OmpF and ompF mRNA after an increase in temperature indicated that multicopies of micF RNA markedly inhibited OmpF synthesis but did not accentuate ompF mRNA decrease. These data suggest that multicopy micF inhibits OmpF synthesis primarily through translational inactivation of ompF mRNA and that a limiting factor in addition to micF RNA is necessary to destabilize ompF mRNA.

Association of higher molecular weight ribonucleoproteins of 7 S particle preparations with multimers of transcription factor IIIA.

High molecular weight (HMW) fractions of Xenopus laevis 7 S ribonucleoprotein (RNP) particle preparations were analyzed for RNA and protein content. RNA/protein ratios, amino acid analyses and Western blots reveal that the major HMW fraction from a non-denaturing polyacrylamide gel (band b) contains two molecules of transcription factor protein IIIA (TFIIIA) to one 5 S RNA. Another HMW band appears to contain 4 molecules of TFIIIA to one 5 S RNA. Yet another RNP band (band a) contains 5 S RNA and a protein unrelated to TFIIIA. Thus, native 7 S particle preparations contain 5 S RNA bound to multimeric forms of TFIIIA as well as to an unrelated protein. The presence of additional TFIIIA molecules associated with 7 S particles may have significance in the sequestering of TFIIIA during transcriptional regulation of the 5 S gene.

Unusual 5 S ribosomal RNAs. An analysis of individual segments can reveal phylogenetic relatedness.

Sequence comparisons of 5 S and other ribosomal RNAs by segments can be useful in understanding anomalous primary and secondary structures and in assessing phylogenetic relationships. In a segmented analysis, the 5'-half of the Chlamydomonas reinhardii chloroplast 5 S ribosomal RNA is found to have a very close sequence homology to the green plant chloroplast and cyanobacterial 5 S RNAs; however, the 3'-half has a highly unusual sequence. Further comparisons of homologies between regions of the 5 S RNAs from C. reinhardii and the green plant chloroplasts suggest that genetic rearrangements within the 5 S DNA may have produced the unusual sequence at the 3'-half. Segmented analyses of the C. reinhardii and green plant chloroplast 5 S RNAs suggest a close relationship which is not revealed by overall sequence comparisons.

The isolation and characterization of RNA coded by the micF gene in Escherichia coli.

A new species of micF RNA, which contains 93 nucleotides (a 4.5S size), was isolated from Escherichia coli. The sequence of the 4.5S micF RNA corresponds to positions G82 through U174 of the micF gene. The 5' terminal end of this smaller micF RNA is triphosphorylated signifying that it is a primary transcript. Its promoter region, which is situated within the greater micF structural gene, has been identified and characterized by lacZ fusion analysis. A 6S micF RNA species, which has a base composition predicted for a transcript from the full length gene has also been detected; however, the 4.5S micF RNA is the predominant species. The work clearly shows by biochemical identification the presence of chromosomally encoded micF RNA.

Origins of the plant chloroplasts and mitochondria based on comparisons of 5S ribosomal RNAs.

In this paper, we provide macromolecular comparisons utilizing the 5S ribosomal RNA structure to suggest extant bacteria that are the likely descendants of chloroplast and mitochondria endosymbionts. The genetic stability and near universality of the 5S ribosomal gene allows for a useful means to study ancient evolutionary changes by macromolecular comparisons. The value in current and future ribosomal RNA comparisons is in fine tuning the assignment of ancestors to the organelles and in establishing extant species likely to be descendants of bacteria involved in presumed multiple endosymbiotic events.

Interrelatedness of 5S RNA sequences investigated by correspondence analysis.

Correspondence analysis (a form of multivariate statistics) applied to 74 5S ribosomal RNA sequences indicates that the sequences are interrelated in a systematic, nonrandom fashion. Aligned sequences are represented as vectors in a 5N-dimensional space, where N is the number of base positions in the 5S RNA molecule. Mutually orthogonal directions (called factor axes) along which intersequence variance is greatest are defined in this hyperspace. Projection of the sequences onto planes defined by these factorial directions reveals clustering of species that is suggestive of phylogenetic relationships. For each factorial direction, correspondence analysis points to regions of "importance," i.e., those base positions at which the systematic changes occur that define that particular direction. In effect, the technique provides a rapid determination of group-specific signatures. In several instances, similarities between sequences are indicated that have only recently been inferred from visual base-to-base comparisons. These results suggest that correspondence analysis may provide a valuable starting point from which to uncover the patterns of change underlying the evolution of a macromolecule, such as 5S RNA.

Characterization of RNA-protein interactions in 7 S ribonucleoprotein particles from Xenopus laevis oocytes.

5 S RNA interactions with transcription factor protein A (TFIIIA) in 7 S particles from Xenopus laevis oocytes (Xlo) have been characterized by the use of an in vitro RNA exchange assay. 32P-labeled Xlo 5 S RNA can rapidly be incorporated into 7 S particles by simple incubation of the RNA with intact particles. Incorporation of the labeled RNA during exchange reaches an equilibrium within 20 min at 20 degrees C. Labeled Xlo 5 S RNA already incorporated in 7 S particles can be chased out by an excess of unlabeled 5 S RNA. Nondenaturing gel electrophoresis of 7 S particle samples segregates several ribonucleoprotein particles containing TFIIIA and 5 S RNA. Time course experiments reveal incorporation of 32P-labeled 5 S RNA first in a higher molecular weight ribonucleoprotein particle before incorporation into the 7 S particle. In the exchange process, the integrity of the higher order structure of the RNA is essential for a recognition of the 5 S RNA by TFIIIA. Denatured Xlo 5 S RNA exchanges poorly in the presence of EDTA, but can exchange into the particle at a high level if sufficient divalent cations are present to allow the higher order structure of the RNA to reform. Xlo 5 S RNA fragments that have the 5' or 3' ends deleted past helix I markedly lose their ability to exchange. Heterologous eukaryotic and eubacterial 5 S RNAs can exchange into 7 S particles, although the eubacterial 5 S RNAs exchange at a low level.