Selection Effects on the Positioning of Genes and Gene Structures from the Interplay of Replication and Transcription in Bacterial Genomes

Bacterial chromosomes are partly shaped by the functional requirements for efficient replication, which lead to strand bias as commonly characterized by the excess of guanines over cytosines in the leading strand. Gene structures are also highly organized within bacterial genomes as a result of such functional constraints, displaying characteristic positioning and structuring along the genome. Here we analyze the gene structures in completely sequenced bacterial chromosomes to observe the positional constraints on gene orientation, length, and codon usage with regard to the positions of replication origin and terminus. Selection on these gene features is different in regions surrounding the terminus of replication from the rest of the genome, but the selection could be either positive or negative depending on the species, and these positional effects are partly attributed to the A-T enrichment near the terminus. Characteristic gene structuring relative to the position of replication origin and terminus is commonly observed among most bacterial species with circular chromosomes, and therefore we argue that the highly organized gene positioning as well as the strand bias should be considered for genomics studies of bacteria

Conservation of the links between gene transcription and chromosomal organization in the highly reduced genome of Buchnera aphidicola

<p>Abstract</p> <p>Background</p> <p>Genomic studies on bacteria have clearly shown the existence of chromosomal organization as regards, for example, to gene localization, order and orientation. Moreover, transcriptomic analyses have demonstrated that, in free-living bacteria, gene transcription levels and chromosomal organization are mutually influenced. We have explored the possible conservation of relationships between mRNA abundances and chromosomal organization in the highly reduced genome of <it>Buchnera aphidicola</it>, the primary endosymbiont of the aphids, and a close relative to <it>Escherichia coli</it>.</p> <p>Results</p> <p>Using an oligonucleotide-based microarray, we normalized the transcriptomic data by genomic DNA signals in order to have access to inter-gene comparison data. Our analysis showed that mRNA abundances, gene organization (operon) and gene essentiality are correlated in <it>Buchnera </it>(i.e., the most expressed genes are essential genes organized in operons) whereas no link between mRNA abundances and gene strand bias was found. The effect of <it>Buchnera </it>genome evolution on gene expression levels has also been analysed in order to assess the constraints imposed by the obligate symbiosis with aphids, underlining the importance of some gene sets for the survival of the two partners. Finally, our results show the existence of spatial periodic transcriptional patterns in the genome of <it>Buchnera</it>.</p> <p>Conclusion</p> <p>Despite an important reduction in its genome size and an apparent decay of its capacity for regulating transcription, this work reveals a significant correlation between mRNA abundances and chromosomal organization of the aphid-symbiont <it>Buchnera</it>.</p

Chromosomal periodicity and positional networks of genes in Escherichia coli

Escherichia coli periodic gene distribution is identified for a periodic interval of 33 kb.Two positional networks of genes are discovered by studying gene periodic distribution: one is driven by metabolic genes and the other by genes involved in cellular processing and signaling.A functional core of Escherichia coli genes drives gene periodic distribution.A few chromosomal regions that preserve gene transcription profiles across environmental changes are identified.This single genome analysis approach can be taken as a footprint for a large-scale bacterial and archaeal periodic distribution analysis

Co-Orientation of Replication and Transcription Preserves Genome Integrity

In many bacteria, there is a genome-wide bias towards co-orientation of replication and transcription, with essential and/or highly-expressed genes further enriched co-directionally. We previously found that reversing this bias in the bacterium Bacillus subtilis slows replication elongation, and we proposed that this effect contributes to the evolutionary pressure selecting the transcription-replication co-orientation bias. This selection might have been based purely on selection for speedy replication; alternatively, the slowed replication might actually represent an average of individual replication-disruption events, each of which is counter-selected independently because genome integrity is selected. To differentiate these possibilities and define the precise forces driving this aspect of genome organization, we generated new strains with inversions either over ∼1/4 of the chromosome or at ribosomal RNA (rRNA) operons. Applying mathematical analysis to genomic microarray snapshots, we found that replication rates vary dramatically within the inverted genome. Replication is moderately impeded throughout the inverted region, which results in a small but significant competitive disadvantage in minimal medium. Importantly, replication is strongly obstructed at inverted rRNA loci in rich medium. This obstruction results in disruption of DNA replication, activation of DNA damage responses, loss of genome integrity, and cell death. Our results strongly suggest that preservation of genome integrity drives the evolution of co-orientation of replication and transcription, a conserved feature of genome organization

Development of an In vitro, coupled transcription-to-translation system for analysis of the interactions between the ribosome and RNA polymerase

PhD ThesisThe various properties of actively transcribing RNA polymerase (RNAP) complexes with nucleic acids during different stages involve various types of regulation and different cross-talk with other cellular entities and with RNAP itself. For instance, transcription and translation are coupled in bacteria, meaning that translation takes place co-transcriptionally. The interactions of transcriptional apparatus with the translational machinery have been focused mainly in terms of gene expression, whereas the study of the physical interaction of the ribosome and the RNA polymerase remains obscure due to the lack of a system which allows such observations. In this study we have developed a pure, transcription-coupled-to-translation system in which the translocation of the ribosome can be performed in a step-wise manner towards RNAP allowing the observation of the outcomes of the interactions between the two machineries at colliding and non-colliding distances; the system also allows the positioning of RNAP in any desired elongation complex such as paused, roadblocked, backtracked, etc. We show the study of the interactions of the ribosome on different aspects of transcription elongation and also the effects on translation caused by RNAP

Development of an in vitro, coupled transcription-to-translation system for analysis of the interactions between the ribosome and RNA polymerase

The various properties of actively transcribing RNA polymerase (RNAP) complexes with nucleic acids during different stages involve various types of regulation and different cross-talk with other cellular entities and with RNAP itself. For instance, transcription and translation are coupled in bacteria, meaning that translation takes place co-transcriptionally. The interactions of transcriptional apparatus with the translational machinery have been focused mainly in terms of gene expression, whereas the study of the physical interaction of the ribosome and the RNA polymerase remains obscure due to the lack of a system which allows such observations. In this study we have developed a pure, transcription-coupled-to-translation system in which the translocation of the ribosome can be performed in a step-wise manner towards RNAP allowing the observation of the outcomes of the interactions between the two machineries at colliding and non-colliding distances; the system also allows the positioning of RNAP in any desired elongation complex such as paused, roadblocked, backtracked, etc. We show the study of the interactions of the ribosome on different aspects of transcription elongation and also the effects on translation caused by RNAP.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Mechanisms and Factors Promoting Faithful Replication of Problematic Genomic Regions

Natural Pausing Sites (NPSs) are complex genomic regions predisposed to fragility. Our lab previously uncovered that the Smc5/6 complex is critical for replication through NPSs. We hypothesized that Smc5/6 maintains NPS integrity by coupling replication fork pausing at different repeat elements with recombination and recombination intermediate resolution. The Sgs1-Top3-Rmi1 (STR) complex and Smc5/6 co-localize genome-wide in G2/M, and prevent accumulation of recombination structures at damaged forks. Here we use several genome-wide and locus-specific methods to investigate the mechanisms and factors involved in NPS metabolism. We find evidence that Smc5/6 collaborates tightly with STR and coordinates various resolvases at NPSs to support replication completion. Smc5/6 chromatin clusters overlap with the ones of Top3 and Rmi1 and are enriched at NPSs, where Smc5/6 facilitates Top3 retention. Further, we observe that Smc6 mutants that accumulate recombination intermediates at replication termination regions encode variants that bind less efficiently to NPSs. Both Smc5/6 dysfunction and STR depletion cause accumulation of recombination intermediates at stalled NPSs. A newly discovered intragenic mutation of smc6-56 restores Top3 binding but causes additivity with various sgs1 mutants, suggesting defects in other resolvases, possibly Mus81-Mms4. We further observe a role for Smc5/6, STR and DNA damage tolerance (DDT) pathways mediated by the polymerase clamp PCNA at topologically constrained regions along with Top2. We observe aggravated top2-4 temperature sensitivity for mutants of the above-mentioned factors, which is independent of Rad51 dependent recombination. Taken together, our results indicate a role for the STR complex in collaboration with Smc5/6 in NPS maintenance by resolving recombination intermediates to allow faithful segregation of these complex genomic regions. We further observe a role for STR, DDT and Smc5/6 along Top2 in facilitating resolution of topological stress before and during mitosis