Characterization of a single-strand origin, ssoU, required for broad host range replication of rolling-circle plasmids (original) (raw)
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Molecular Microbiology, 2006
A number of small, multicopy plasmids from Gram-positive bacteria replicate by an asymmetric rolling-circle mechanism. Previous studies with several of these plasmids have identified a palindromic sequence, SSOA, that acts as the single-strand origin (SSO) for the replication of the lagging-strand DNA. Although not all the SSOA sequences share ONA sequence homology, they are structurally very similar. We have used an in vitro system to study the lagging-strand replication of several plasmids from Gram-positive bacteria using the SSOA sequences of pT181, pE194 and pSN2 as representative of three different groups of Staphylococcus aureus plasmids. In addition, we have investigated the lagging-strand replication of the pUB110 plasmid that contains an alternative single-strand origin, ssou. Our results confirm that RNA polymerase is involved in lagging-strand synthesis from both SSOA and ssou-type lagging-strand origins. Interestingly, while initiation of lagging-strand DNA synthesis of pUB110 occurred predominantly at a single position within ssou, replication of pT181, pSN2 and pE194 plasmids initiated at multiple positions from SSOA.
Journal of Bacteriology, 2000
Adaptation of bacterial cells to diverse habitats relies on the ability of RNA polymerase to respond to various regulatory signals. Some of these signals are conserved throughout evolution, whereas others are species specific. In this study we present a comprehensive comparative analysis of RNA polymerases from two distantly related bacterial species, Escherichia coli and Bacillus subtilis, using a panel of in vitro transcription assays. We found substantial species-specific differences in the ability of these enzymes to escape from the promoter and to recognize certain types of elongation signals. Both enzymes responded similarly to other pause and termination signals and to the general E. coli elongation factors NusA and GreA. We also demonstrate that, although promoter recognition depends largely on the subunit, promoter discrimination exhibited in species-specific fashion by both RNA polymerases resides in the core enzyme. We hypothesize that differences in signal recognition are due to the changes in contacts made between the  and  subunits and the downstream DNA duplex. on June 23, 2015 by guest http://jb.asm.org/ Downloaded from on June 23, 2015 by guest http://jb.asm.org/ Downloaded from on June 23, 2015 by guest http://jb.asm.org/ Downloaded from
Escherichia coli and Pseudomonas putida RNA polymerases display identical contacts with promoters
Molecular and General Genetics, 1984
Methylation protection experiments with four promoters (P1 and P2 of the pBR322 plasmid, lacUV5 and lambda P0) have shown that the RNA polymerases from Escherichia coli and Pseudomonas putida, while differing in the primary structure of the subunits involved in DNA binding, display identical patterns of DNA contacts. Nor do these enzymes differ in covalent cross-linking patterns with a partially apurinized promoter. We conclude that the two RNA polymerases have very similar structures of DNA binding centers. The evolutionary conservation of this structure may account for the fact that diverse RNA polymerases often recognize and efficiently use promoters of distant bacterial species.
Methods in Molecular Biology - E. coli Plasmid Vectors
ColE1 uses an extensive RNA primer for leading-strand synthesis. The RNAII preprimer is transcribed from the RNAII promoter by host RNA polymerase. RNAII forms a persistent RNA-DNA hybrid at the plasmid origin of replication. This hybrid is cleaved by RNase H and the resulting free 3'OH group on the cleaved RNAII acts as a primer for continuous leading-strand synthesis, catalyzed by host DNA polymerase I. ColE1 regulates its copy number with a short RNA countertranscript, RNAI. This species is expressed constitutively from the strong RNAI promoter, is nontranslated, and is fully complementary to part of RNAII. The interaction of RNAI with RNAII results in an RNAII configuration that impairs further elongation of this transcript, thereby reducing the frequency of RNA-DNA duplex formation and initiation of replication. The RNAI-RNAII interaction is counterbalanced by the shorter half-life of RNAI compared to RNAII. The ColE1-encoded Rom protein (also known as Rop) increases the frequency of RNAI-RNAII interactions. The gene for Rom is deleted in many ColE1-based plasmid vectors, resulting in increased copy numbers compared to ColE1 itself. Perturbations of ColE1 plasmid copy number are rapidly mirrored by changes in RNAI concentration, resulting in the enhancement or suppression of replication and the maintenance of ColE1 copy number within a narrow window. 4.3. Rolling-Circle Replication Many small (<10 kbp) plasmids of Gram-positive Eubacteria replicate by a rollingcircle mechanism, which is distinct from the replication of iteron-containing or ColE1like plasmids (see Fig. 3) (47). Rolling-circle plasmids have also been identified in Gram-negative Eubacteria and in Archaea. Some bacteriophage, including M13 of E. coli, also replicate in this way. In rolling-circle replication, binding of a plasmid-encoded replication protein to the leading-strand origin (also known as the double-strand origin) distorts the DNA in this region and exposes a single-stranded region in an extruded cruciform. A nick is introduced at this site by the replication protein and this exposes a 3'OH group from which the leading strand is synthesized by DNA polymerase III. Leading strand initiation differs between rolling circle plasmids, procaryotic chromosomes, and other plasmids, although chain elongation is similar in all systems. As the leading strand is synthesized, the nontemplate strand of the old plasmid is displaced ahead of the replication fork until, eventually, it is removed entirely. The resulting single-stranded intermediate is characteristic of rolling-circle replication and its identification provides evidence that a plasmid replicates by this mechanism (48). The lagging-strand origin (also known as the single-strand origin) is exposed on the displaced single-stranded intermediate and lagging-strand initiation commences at this origin using host replication factors. RNA polymerase synthesizes RNA primers at the lagging strand origin. DNA polymerase I initiates lagging strand synthesis from these RNA primers, after which DNA polymerase III continues elongation. 5. Plasmid Segregation DNA replication produces precise plasmid copies, but plasmids must also ensure that they are distributed to both daughter cells during bacterial cell division. If the Hayes
Journal of Biological Chemistry, 2007
2 The abbreviations used are: RNAP, holo-RNA polymerase (if not further specified, the factor is WT 70 ); FYWW RNAP, RNA polymerase containing 70 with four substitutions (F427A, Y430A, W433A, and W434A); WT, wild type; MES, 4-morpholineethanesulfonic acid; CRP, cyclic AMP receptor protein.
Nucleic Acids Research, 2011
Specific promoter recognition by bacterial RNA polymerase is mediated by p subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, p 70 (the housekeeping p subunit) and p S (an alternative p subunit mostly active during slow growth) recognize almost identical promoter sequences, thus raising the question of how promoter selectivity is achieved in the bacterial cell. To identify novel sequence determinants for selective promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA polymerase saturated either with p 70 (Ep 70) or with p S (Ep S) using the whole Escherichia coli genome as DNA template. We found that Ep 70 , in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, Ep S efficiently transcribes genes involved in stress responses, secondary metabolism as well as RNAs from intergenic regions with yet-unknown function. Promoter sequence comparison suggests that, in addition to different conservation of the À35 sequence and of the UP element, selective promoter recognition by either form of RNA polymerase can be affected by the A/ T content in the À10/+1 region. Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the À10/+1 region could improve promoter recognition by Ep S .
Journal of Bacteriology, 2002
Sinorhizobium meliloti, a gram-negative soil bacterium, forms a nitrogen-fixing symbiotic relationship with members of the legume family. To facilitate our studies of transcription in S. meliloti, we cloned and characterized the gene for the ␣ subunit of RNA polymerase (RNAP). S. meliloti rpoA encodes a 336-amino-acid, 37-kDa protein. Sequence analysis of the region surrounding rpoA identified six open reading frames that are found in the conserved gene order secY (SecY)-adk (Adk)-rpsM (S13)-rpsK (S11)-rpoA (␣)-rplQ (L17) found in the ␣-proteobacteria. In vivo, S. meliloti rpoA expressed in Escherichia coli complemented a temperature sensitive mutation in E. coli rpoA, demonstrating that S. meliloti ␣ supports RNAP assembly, sequence-specific DNA binding, and interaction with transcriptional activators in the context of E. coli. In vitro, we reconstituted RNAP holoenzyme from S. meliloti ␣ and E. coli , , and subunits. Similar to E. coli RNAP, the hybrid RNAP supported transcription from an E. coli core promoter and responded to both upstream (UP) elementand Fis-dependent transcription activation. We obtained similar results using purified RNAP from S. meliloti. Our results demonstrate that S. meliloti ␣ functions are conserved in heterologous host E. coli even though the two ␣ subunits are only 51% identical. The ability to utilize E. coli as a heterologous system in which to study the regulation of S. meliloti genes could provide an important tool for our understanding and manipulation of these processes.