512 research outputs found
The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly
Escherichia coli RNase E, an essential single-stranded specific endoribonuclease, is required for both ribosomal RNA processing and the rapid degradation of mRNA. The availability of the complete sequences of a number of bacterial genomes prompted us to assess the evolutionarily conservation of bacterial RNase E. We show here that the sequence of the N-terminal endoribonucleolytic domain of RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria. Furthermore, we demonstrate that the Synechocystis sp. homologue binds RNase E substrates and cleaves them at the same position as the E. coli enzyme. Taken together these results suggest that RNase E-mediated mechanisms of RNA decay are not confined to E. coli and its close relatives. We also show that the C-terminal half of E. coli RNase E is both sufficient and necessary for its physical interaction with the 3'-5' exoribonuclease polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase, which are components of a "degradosome" complex. Interestingly, however, the sequence of the C-terminal half of E. coli RNase E is not highly conserved evolutionarily, suggesting diversity of RNase E interactions with other RNA decay components in different organisms. This notion is supported by our finding that the Synechocystis sp. RNase E homologue does not function as a platform for assembly of E. coli degradosome components
The Conserved Dcw Gene Cluster of R. sphaeroides Is Preceded by an Uncommonly Extended 5Ā Leader Featuring the sRNA UpsM
Cell division and cell wall synthesis mechanisms are similarly conserved among bacteria. Consequently some bacterial species have comparable sets of genes organized in the dcw (division and cell wall) gene cluster. Dcw genes, their regulation and their relative order within the cluster are outstandingly conserved among rod shaped and gram negative bacteria to ensure an efficient coordination of growth and division. A well studied representative is the dcw gene cluster of E. coli. The first promoter of the gene cluster (mraZ1p) gives rise to polycistronic transcripts containing a 38 nt long 5Ā UTR followed by the first gene mraZ. Despite reported conservation we present evidence for a much longer 5Ā UTR in the gram negative and rod shaped bacterium Rhodobacter sphaeroides and in the family of Rhodobacteraceae. This extended 268 nt long 5Ā UTR comprises a Rho independent terminator, which in case of termination gives rise to a non-coding RNA (UpsM). This sRNA is conditionally cleaved by RNase E under stress conditions in an Hfq- and very likely target mRNA-dependent manner, implying its function in trans. These results raise the question for the regulatory function of this extended 5Ā UTR. It might represent the rarely described case of a trans acting sRNA derived from a riboswitch with exclusive presence in the family of Rhodobacteraceae
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Directed evolution of antimutator E. coli
Biological systems are essential tools for addressing societal challenges. There have been several successes in this field, however, a strong hindrance lies in the ephemeral nature of these systems ā cells are tiny factories that evolve. Evolution poses a problem because when a desired function is encoded into the DNA of the host organism, the host uses its own resources to perform the function and there is likely no associated fitness benefit. Therefore, there is strong selection for inactivation of the function due to the metabolic load imposed on cellular resources. One way to address this problem is to engineer evolutionary stability by lowering a host organismās basal mutation rate and concomitantly reducing the probability that an encoded function will become mutated.
In Chapter 1 of this dissertation, I discuss the nature of the metabolic cost associated with engineering biology and mechanisms by which host adaptation occurs. I also explore cellular pathways involved in genetic stability and examine previously characterized antimutators. Chapter 2 describes the first iteration of a directed evolution method used to engineer antimutators in Escherichia coli, Periodic Reselection for Evolutionarily Reliable Variants (PResERV). In this first PResERV experiment, I observe that the antimutator phenotype is due to mutations in genes involved DNA replication and RNA metabolism (polA, polB, and rne). In Chapter 3, I perform the same PResERV experiment on a greater scale and characterize a series of antimutator strains. The causative alleles in many of these strains are in genes involved in the tricarboxylic acid cycle and electron transport chain (sucD and sdhA). These alleles are shown to reduce oxidative stress. Chapter 4 demonstrates results from another PResERV experiment using a clean-genome E. coli strain, MDS42, as the host organism. In sum, this work shows the many mechanisms that lead to an antimutator phenotype, and these findings are used to build stable strains for reliable engineering of biology.
Finally, there are two appendices (Appendix A and B) which discuss my work in examining the evolutionary path to citrate utilization in Lenskiās long-term evolution experiment (LTEE) and a do-it-yourself method for using gellan gum as an alternative to microbial agar media.Microbiolog
Evolution of Escherichia coli to 42 Ā°C and Subsequent Genetic Engineering Reveals Adaptive Mechanisms and Novel Mutations.
Adaptive laboratory evolution (ALE) has emerged as a valuable method by which to investigate microbial adaptation to a desired environment. Here, we performed ALE to 42 Ā°C of ten parallel populations of Escherichia coli K-12 MG1655 grown in glucose minimal media. Tightly controlled experimental conditions allowed selection based on exponential-phase growth rate, yielding strains that uniformly converged toward a similar phenotype along distinct genetic paths. Adapted strains possessed as few as 6 and as many as 55 mutations, and of the 144 genes that mutated in total, 14 arose independently across two or more strains. This mutational recurrence pointed to the key genetic targets underlying the evolved fitness increase. Genome engineering was used to introduce the novel ALE-acquired alleles in random combinations into the ancestral strain, and competition between these engineered strains reaffirmed the impact of the key mutations on the growth rate at 42 Ā°C. Interestingly, most of the identified key gene targets differed significantly from those found in similar temperature adaptation studies, highlighting the sensitivity of genetic evolution to experimental conditions and ancestral genotype. Additionally, transcriptomic analysis of the ancestral and evolved strains revealed a general trend for restoration of the global expression state back toward preheat stressed levels. This restorative effect was previously documented following evolution to metabolic perturbations, and thus may represent a general feature of ALE experiments. The widespread evolved expression shifts were enabled by a comparatively scant number of regulatory mutations, providing a net fitness benefit but causing suboptimal expression levels for certain genes, such as those governing flagellar formation, which then became targets for additional ameliorating mutations. Overall, the results of this study provide insight into the adaptation process and yield lessons important for the future implementation of ALE as a tool for scientific research and engineering
Characterization of the role of ribonucleases in Salmonella small RNA decay
In pathogenic bacteria, a large number of sRNAs coordinate adaptation to stress and expression of virulence genes. To better understand the turnover of regulatory sRNAs in the model pathogen, Salmonella typhimurium, we have constructed mutants for several ribonucleases (RNase E, RNase G, RNase III, PNPase) and Poly(A) Polymerase I. The expression profiles of four sRNAs conserved among many enterobacteria, CsrB, CsrC, MicA and SraL, were analysed and the processing and stability of these sRNAs was studied in the constructed strains. The degradosome was a common feature involved in the turnover of these four sRNAs. PAPI-mediated polyadenylation was the major factor governing SraL degradation. RNase III was revealed to strongly affect MicA decay. PNPase was shown to be important in the decay of these four sRNAs. The stability of CsrB and CsrC seemed to be independent of the RNA chaperone, Hfq, whereas the decay of SraL and MicA was Hfq-dependent. Taken together, the results of this study provide initial insight into the mechanisms of sRNA decay in Salmonella, and indicate specific contributions of the RNA decay machinery components to the turnover of individual sRNAs
The Mechanism and Modulation of H-NS Mediated Repression in Escherichia coli.
The histone-like nucleoid structuring protein H-NS acts as a global repressor of genes that are expressed in response to environmental stimuli and stress conditions. Repression by H-NS is presumably mediated by binding of H NS to primary "nucleation sites" close to promoters, and the formation of extended nucleoprotein complex from these nucleation sites to inhibit transcription initiation. Modulation of H-NS mediated repression is a complex process involving specific transcription factors and physiology dependent structural alterations. The E. coli bgl and proU operons are model systems that are repressed by H-NS with exceptional specificity. Both of these systems possess upstream and downstream regulatory elements (URE and DRE) bound by H-NS for efficient repression. The present study demonstrates that repression by H-NS binding upstream and downstream is synergistic in proU (as shown in a parallel study for bgl), and that H-NS when bound within the transcription unit represses transcription initiation at the bgl promoter, as reported before for proU. Repression by binding of H-NS downstream is known to be modulated. Common to both proU and bgl is that an increase in the promoter activity abrogates repression. For bgl it is known, that the H-NS mediated repression of the promoter is counteracted by transcription factors BglJ and LeuO. Further, termination factor Rho and the protease Lon are known to modulate repression by H-NS through the DRE, and as shown here the DnaKJ chaperone system is essential for this repression. In case of proU, the promoter is osmoregulated; the RNA polymerase is poised at the promoter at low osmolarity, while it clears the promoter with better efficiency at high osmolarity. Furthermore, the proU operon is subject to post-transcriptional osmoregulation. The proU mRNA is processed by RNAse III within a stretch of highly conserved sequence, suggesting a common mechanism of regulation among Enterobacteria. In summary, the present study demonstrates that the mechanism of H-NS mediated repression of the bgl and proU operons is very similar. However, its modulation is complex involving numerous additional factors specific to the two systems, and thus is achieved in a context specific manner
Direct entry by RNase E is a major pathway for the degradation and processing of RNA in Escherichia coli
Escherichia coli endoribonuclease E has a major influence on gene expression. It is essential for the maturation of ribosomal and transfer RNA as well as the rapid degradation of messenger RNA. The latter ensures that translation closely follows programming at the level of transcription. Recently, one of the hallmarks of RNase E, i.e. its ability to bind via a 5'-monophosphorylated end, was shown to be unnecessary for the initial cleavage of some polycistronic tRNA precursors. Here we show using RNA-seq analyses of ribonuclease-deficient strains in vivo and a 5'-sensor mutant of RNase E in vitro that, contrary to current models, 5'-monophosphate-independent, 'direct entry' cleavage is a major pathway for degrading and processing RNA. Moreover, we present further evidence that direct entry is facilitated by RNase E binding simultaneously to multiple unpaired regions. These simple requirements may maximize the rate of degradation and processing by permitting multiple sites to be surveyed directly without being constrained by 5'-end tethering. Cleavage was detected at a multitude of sites previously undescribed for RNase E, including ones that regulate the activity and specificity of ribosomes. A potentially broad role for RNase G, an RNase E paralogue, in the trimming of 5'-monophosphorylated ends was also revealed
Rho-independent transcription terminators inhibit RNase P processing of the secG leuU and metT tRNA polycistronic transcripts in Escherichia coli
The widely accepted model for the processing of tRNAs in Escherichia coli involves essential initial cleavages by RNase E within polycistronic transcripts to generate pre-tRNAs that subsequently become substrates for RNase P. However, recently we identified two polycistronic tRNA transcripts whose endonucleolytic processing was solely dependent on RNase P. Here we show that the processing of the secG leuU and metT leuW glnU glnW metU glnV glnX polycistronic transcripts takes place through a different type of maturation pathway. Specifically, RNase P separates the tRNA units within each operon following the endonucleolytic removal of the distal Rho-independent transcription terminator, primarily by RNase E. Failure to remove the Rho-independent transcription terminator inhibits RNase P processing of both transcripts leading to a decrease in mature tRNA levels and dramatically increased levels of full-length transcripts in an RNase E deletion strain. Furthermore, we show for the first time that RNase G also removes the Rho-independent transcription terminator associated with the secG leuU operon. Our data also demonstrate that the Rne-1 protein retains significant activity on tRNA substrates at the non-permissive temperature. Taken together it is clear that there are multiple pathways involved in the maturation of tRNAs in E. coli
A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis*
Endoribonuclease E (RNase E) is a regulator of global gene expression in Escherichia coli and is the best studied member of the RNase E/G ribonuclease family. Homologues are present in other bacteria but the roles of plant RNase E/G-like proteins are not known. Arabidopsis thaliana contains a single nuclear gene (At2g04270) encoding a product with the conserved catalytic domain of RNase E/G-like proteins. At2g04270 and the adjacent At2g04280 gene form converging transcription units with a ā¼40 base overlap at their 3ā ends. Several translation products were predicted from the analyses of At2g04270 cDNAs. An antibody raised against a recombinant A. thaliana RNase E/G-like protein recognized a 125 kDa protein band in purified chloroplast preparations fractionated by SDS-PAGE. The 125 kDa RNase E/G-like protein was detected in cotyledons, rosette and cauline leaves. T-DNA insertions in exon 6 or intron 11 of At2g04270 result in loss of the 125 kDa band or truncation to a 110 kDa band. Loss of At2g04270 function resulted in the arrest of chloroplast development, loss of autotrophic growth, and reduced plastid ribosomal, psbA and rbcL RNA levels. Homozygous mutant plants were pale-green, contained smaller plastids with fewer thylakoids and shorter granal stacks than wild-type chloroplasts, and required sucrose at all growth stages following germination right up to flowering and setting seeds. Recombinant A. thaliana RNase E/G-like proteins rescued an E. coli RNase E mutant and cleaved an rbcL RNA substrate. Expression of At2g04270 was highly correlated with genes encoding plastid polyribonucleotide phosphorylase, S1 RNA-binding, and CRS1/YhbY domain proteins
Interplay of Exoribonucleases, Hfq and Small RNAs Structural Determinants in the Control of Gene Expression
Dissertation presented to obtain the Ph.D degree in BiologySmall non-coding RNAs (sRNAs) are regulatory molecules that typically are
not translated into proteins. These molecules are often highly structured and very
stable and can affect many genetic pathways in all domains of life. Bacterial small
regulatory RNAs (sRNAs) parallel microRNAs in their ability to control multiple
targets. Small RNAs can bind to proteins or to mRNA targets. The sRNAs that act
by an antisense mechanism can have full (cis-encoded) or partial complementarity
(trans-encoded) with their targets. Most of the trans-encoded sRNAs studied so
far in Escherichia coli bind the RNA chaperone Hfq. The 5ā end of antisense RNAs is
usually found to be critical for the interaction with targets, generally inhibiting
translation and promoting mRNA decay. RNases are key elements in the control of
RNA levels in the cell and not surprisingly are also critical in the regulation of
sRNAs. In E. coli there are three 3ā-5āexoribonucleases that accomplish most of the
mRNA exodegradative activity: ribonuclease II (RNase II), ribonuclease R (RNase R)
and polynucleotide phosphorylase (PNPase).(...
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