Inhibition of ColE1 RNA primer formation by a plasmid-specified small RNA.

Tomizawa, Jun-ichi; Itoh, Tateo; Selzer, Gerald; Som, Tapan

doi:10.1073/pnas.78.3.1421

Cited by 311 publications

(156 citation statements)

References 15 publications

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“…Bacteriophage λ repressor CI (20-1,600 nM) was added to supercoiled DNA templates (4 nM) and the different promoters were transcribed by 20 nM RNA polymerase in the presence of nucleoside 5'-triphosphates and 5 μCi ½α-32 P UTP (1Ci ¼ 37 GBq) (3;000 Ci∕mmol). The RNAI transcripts present in the plasmids (106 and 108 nts) were used as internal controls to quantify the relative amount of transcripts from P R , P L , and P RM (57). The relative amount of transcripts in the presence of CI was normalized to that in the absence of CI.…”

Section: Methodsmentioning

confidence: 99%

Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro

Lewis

Zurla

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The prophage state of bacteriophage λ is extremely stable and is maintained by a highly regulated level of λ repressor protein, CI, which represses lytic functions. CI regulates its own synthesis in a lysogen by activating and repressing its promoter, P RM . CI participates in long-range interactions involving two regions of widely separated operator sites by generating a loop in the intervening DNA. We investigated the roles of each individual site under conditions that permitted DNA loop formation by using in vitro transcription assays for the first time on supercoiled DNA that mimics in vivo situation. We confirmed that DNA loops generated by oligomerization of CI bound to its operators influence the autoactivation and autorepression of P RM regulation. We additionally report that different configurations of DNA loops are central to this regulation-one configuration further enhances autoactivation and another is essential for autorepression of P RM .activation | cooperativity | repression B acteriophage lambda (λ) of Escherichia coli can grow either in a lytic or lysogenic mode. In a lysogenic cell, the phageencoded λ repressor protein (CI) prevents lytic growth by directly repressing two promoters needed to express lytic functions, P L and P R (1-3). Each promoter is associated with a CI recognition site or operator,. O R is associated with promoter P RM (promoter for maintenance of repressor synthesis), which directs transcription of the cI gene in the prophage state (4-6). Each subsite binds a CI dimer (7).The CI protein autoregulates its synthesis. At low cellular CI concentration, CI enhances its own synthesis from P RM ; when high, CI represses P RM (1,8,9). It was originally believed that both positive and negative autoregulations are achieved exclusively by the action of CI dimers at the P RM -O R -P R sequence of the phage genome (1, 10) (Fig. 1A), based on the following observations. (i) There is a hierarchy of intrinsic binding affinities of a CI dimer to individual operators sites: (11)(12)(13)(14)(15)(16)(17); (ii) CI bound to the intrinsically weak O R 2 site is strengthened by cooperative interactions with CI bound to the stronger adjacent O R 1 site, and the ensemble of two CI dimers at the O R 1 ∼ O R 2 sites represses P R and activates P RM (13, 16) (Fig. 1A); (iii) at high CI concentrations, a CI dimer can bind to the weakest operator site, O R 3, repressing P RM (1, 2) (Fig. 1C). Incidentally, a second pair of CI dimers binds cooperatively to O L 1 ∼ O L 2, and represses P L (7).This model was recently modified on the basis of physical and genetic experiments showing that a CI tetramer cooperatively bound to O R 1 ∼ O R 2 interacts with a tetramer cooperatively bound to O L 1 ∼ O L 2, located 2.3 kbp away (18-21), resulting in the looping out of the intervening DNA, as shown by electron and atomic force microscopy (18,22,23). DNA loop was also observed by cooperative interactions of CI at sites separated by only five and six helical turns (23, 24). The kinetic and thermodynamic properties ...

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Section: Methodsmentioning

confidence: 99%

Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro

Lewis

Zurla

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…They act by binding to and inhibiting the function of a target RNA. Two Escherichia coli systems that have been well characterized are the IncFII plasmids, in which the antisense RNA, CopA, interacts with its mRNA target to inhibit translation of an essential replication initiator protein (Nordström et al, 1984;Blomberg et al, 1994;Malmgren et al, 1996), and the ColE1-related plasmids, in which the antisense RNA, RNA I, prevents the maturation of a preprimer RNA (Tomizawa et al, 1981). There is considerable similarity in the way in which these systems work.…”

Section: Introductionmentioning

confidence: 99%

Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA

Söderbom

Binnie

Masters

et al. 1997

Molecular Microbiology

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SummaryThe replication frequency of plasmid R1 is controlled by an unstable antisense RNA, CopA, which, by binding to its complementary target, blocks translation of the replication rate-limiting protein RepA. Since the degree of inhibition is directly correlated with the intracellular concentration of CopA, factors affecting CopA turnover can also alter plasmid copy number. We show here that PcnB (PAP I -a poly(A)polymerase of Escherichia coli ) is such a factor. Previous studies have shown that the copy number of ColE1 is decreased in pcnB mutant strains because the stability of the RNase E processed form of RNAI, the antisense RNA regulator of ColE1 replication, is increased. We find that, analogously, the twofold reduction in R1 copy number caused by a pcnB lesion is associated with a corresponding increase in the stability of the RNase E-generated 3Ј cleavage product of CopA. These results suggest that CopA decay is initiated by RNase E cleavage and that PcnB is involved in the subsequent rapid decay of the 3Ј CopA stem-loop segment. We also find that, as predicted, under conditions in which CopA synthesis is unaffected, pcnB mutation reduces RepA translation and increases CopA stability to the same extent.

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“…The DNA region between the pla and pst genes has high similarity (> 70%) to the intergenic region which is located upstream of colicin 10 (Pilsl & Braun, 1995). Another stretch of DNA showing high homology to RNA I of the ColEl plasmid (Morita & Oka, 1979;Tomizawa et al, 1981) can be identified upstream of the pim gene, bp2501-2612. The presence of an RNA I encoding sequence which is highly homologous to RNA I of colicinogenic plasmid ColEl defines the Col-type origin of replication of the pYP358 plasmid.…”

Section: Lysis Peptide and Other Possible Orfsmentioning

confidence: 99%

Structural and functional organization of the Yersinia pestis bacteriocin pesticin gene cluster

Rakin¹,

Boolgakowa

Heesemann³

1996

Microbiology

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The primary structure of a 2671 bp DNA fragment between the pla gene (encoding plasminogen activator) and the origin of replication of the wild-type Yersinia pestis plasmid pYP358 was determined. Two ORFs of 1074 and 426 bp with opposite transcription polarities were identified on both strands. They encode a 357 aa pesticin activity protein (Pst) and a 141 aa pesticin immunity polypeptide (Pim). A GC-rich palindromic structure located between pst and pim can form a hairpin loop and serve as a rho-independent transcription terminator sequence for both genes. The site for the interaction with the LexA repressor of the SOS system was found in another palindromic structure preceding the pst structural gene. A deduced 399 kDa Pst polypeptide is devoid of a signal peptide, indicating a Sec-independent mode of export. Pst carries a pentapeptide typical of TonB-dependent colicins (TonB box) that is necessary for the interaction with the yersiniabactidpesticin receptor and for active energyldependent transport through the outer membrane. The substitution of the last five C-terminal amino acids did not significantly influence the bactericidal activity of the truncated pesticin. The pesticin lost its ability to kill sensitive bacteria and to bind to a pesticin receptor after deletion of the last 57 C-terminal amino acids. A deduced 16 kDa Pim protein has an Nterminal hydrophobic amino acid stretch with features typical of prokaryotic signal peptides. Pim is a slightly hydrophilic protein with a basic pl. The immunity protein was localized in the periplasmic space and in the outermembrane fraction after its overexpression under the polymerase T7 promoter. Several other ORFs were identified on the sequenced 2671 bp fragment, but none of them seemed to encode a typical lysis peptide, which is necessary for the release of the pesticin. In the promoter region and in the regions preceding and following the pst operon, the DNA sequence has high (> 70%) identity with other colicin genes. The DNA sequence located 284 bp upstream of the pim gene showed more than 90% similarity to antisense RNA I of the ColEl replicon. This defined the location of the pYP358 origin of ColE1-type replication.

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Inhibition of ColE1 RNA primer formation by a plasmid-specified small RNA.

Cited by 311 publications

References 15 publications

Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro

Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro

Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA

Structural and functional organization of the Yersinia pestis bacteriocin pesticin gene cluster

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