Transcription of the replication control region of the IncFII R-plasmid NR1 in Vitro and in Vivo

Womble, David D.; Sampathkumar, P.; Easton, Alan M.; Luckow, Verne A.; Rownd, Robert H.

doi:10.1016/0022-2836(85)90228-1

Cited by 37 publications

(53 citation statements)

References 39 publications

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“…First, there was a residual, or basal, level of expression of repAl when leader peptide translation was inhibited either by the high concentration of RNA-E from the wild-type fusion plasmid or by the nonsense mutation in the leader peptide gene of the PE mutant fusion plasmid. Second, the approximate twofold increase in the basal level of expression of repAl in the PE mutant probably resulted from the twofold increased rate of transcription of repAl mRNA in the absence of interference from convergent RNA-E transcription, as previously observed (44). In a finding consistent with the pattern for low-copy-number translational-fusion plasmids, the repA2 mutation, by itself or in combination with the PE mutation, increased the expression of both the leader peptide and repAl (Table 3).…”

Section: Resultssupporting

confidence: 62%

“…Alternatively, the secondary structure of the leader mRNA might be affected in a way that inhibits ribosome binding, by a mechanism similar to that proposed earlier for repAl mRNA (9,40). RNA-E is transcribed from promoter PE, which is a constitutive promoter about 20 times stronger than Pc (44). Therefore, RNA-E is provided in proportion to plasmid copy number and is in excess compared with its target sequences in repAl mRNA (44).…”

Section: Discussionmentioning

confidence: 90%

“…When PA is repressed, there is a low level of constitutive transcription of repAl that is initiated at promoter Pc (8,44). Direct measurements of the rates of RNA transcription indicated that PA is about six times stronger than Pc, so that derepression of PA causes a significant increase in the rate of transcription of repAl mRNA (44). The repA2 gene is transcribed constitutively and provides RepA2 repressor protein in proportion to plasmid copy number (8).…”

Section: Discussionmentioning

confidence: 99%

“…1). Transcription of repAl mRNA may be initiated from a constitutive promoter, Pc, and a regulated promoter, PA, to synthesize RNA-CX and RNA-A, respectively (13,31,44). RNA-CX also serves as the mRNA for the repA2 gene, peptide (45).…”

mentioning

confidence: 99%

“…Regulation of translation of repAl mRNA by RNA-E may occur indirectly by MATERIALS AND METHODS Bacterial strains and culture conditions. E. coli K-12 strain JG112 met thy rpsL polA (23) and its polA+ revertant JK17 (44) were used for replicon reconstitution experiments, for testing thepoL4 dependence of chimeric plasmids composed of both pBR322 and NR1 replicons, and for plasmid copy number and stability assays. E. coli MC1061 araDJ39 A(araleu)697 AlacX74 galU galK rpsL hsdR (6) was used for construction of lacZ fusion plasmids.…”

mentioning

confidence: 99%

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Suppression of replication-deficient mutants of IncFII plasmid NR1 can occur by two different mechanisms that increase expression of the repA1 gene

Wang

Womble

et al. 1993

J Bacteriol

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Replication-proficient (Rep') revertants were isolated from mutants of IncFll plasmid NR1 that were replication defective (Rep-). The parental Rep-plasmids contained a mutation that inactivated promoter PE for transcription of RNA-E, a trans-acting repressor of translation of the essential RepAl replication initiation protein of NR1. The PE mutation also introduced a nonsense codon into a leader peptide gene that precedes and slightly overlaps the repAl translation initiation site in the mRNA. This reduced the rate of synthesis of RepAl by uncoupling its translation from that of the leader peptide. The reduced rate of RepAl synthesis was responsible for the Rep-phenotype. All Rep' revertants retained the PE mutation and contained second-site mutations responsible for suppression of the Rep-phenotype. One Rep+ revertant contained a second mutation adjacent to the Shine-Dalgarno sequence of repAl. Another Rep+ revertant contained a mutation in the repA2 gene, which encodes the trans-acting repressor of transcription of repAl. By using translational JacZ gene fusions, it was found that both kinds of suppressor mutation increased the expression of repAl to a level sufficient to support replication. In both cases, the synthesis of RepAl remained uncoupled from that of the leader peptide. The Shine-Dalgarno mutation increased the rate of leader peptide-independent translation of repAl mRNA and also reduced the sensitivity of repAl mRNA to inhibition by RNA-E. The repA2 mutation inactivated the RepA2 repressor and increased the rate of transcription ofrepAl mRNA. The translational lacZ gene fusions were used to assess the range of regulation of expression of repAl provided by each of the RNA-E and RepA2 regulatory circuits. By constructing miniplasmids that contained various combinations of the mutations, the contributions of the RNA-E and RepA2 regulatory circuits were assessed with respect to control of plasmid copy number and stable inheritance. Plasmids that lacked either circuit were less stable than wild-type plasmids.

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Section: Resultssupporting

confidence: 62%

Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Suppression of replication-deficient mutants of IncFII plasmid NR1 can occur by two different mechanisms that increase expression of the repA1 gene

Wang

Womble

et al. 1993

J Bacteriol

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Control of replication of plasmid R1: translation of the 7k reading frame in the RepA mRNA leader region counteracts the interaction between CopA RNA and CopT RNA.

Wagner¹,

Heijne²,

Nordström³

1987

The EMBO Journal

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Replication of IncFII plasmids is regulated through the expression of a gene, repA. The RepA protein is rate‐limiting for initiation of replication. The main negative control is exerted by a countertranscript, CopA RNA, that binds to the complementary region of the RepA mRNA, thereby inhibiting the formation of the RepA protein. The target region for CopA RNA, CopT, is located upstream of the RepA coding region. An open reading frame for a putative 7k protein overlaps the CopT sequence. Here we show by using lacZ fusions that the 7k gene is expressed. We constructed a translation start mutation in order to abolish formation of the 7k protein. This resulted in a 10‐fold decrease in repA expression. The 7k protein produced in trans did not reverse this effect, so the 7k protein per se does not control expression of repA. However, translation of the 7k coding sequence must influence CopA/CopT RNA recognition, since ribosomes will transiently disrupt the target hairpin. We propose here a novel mechanism that affects the level of gene expression: the 7k region of the RepA mRNA is a leader sequence that is involved in expression of the downstream gene; translation of the 7k region competes with a negative control system involving RNA‐RNA interaction.

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The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain.

et al. 1989

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IS10 transposition is regulated by an approximately 70 nt anti‐sense RNA, RNA‐OUT. RNA‐OUT folds into a duplex ‘stem‐domain’ topped by a loosely paired ‘loop‐domain’. The loop‐domain is critical for RNA‐RNA pairing per se; pairing initiates by interaction of the RNA‐OUT loop with the 5′ end of the target mRNA. We show here that RNA‐OUT is unusually stable in vivo (half‐life 60 min) and that this stability is conferred by specific features of the RNA‐OUT stem‐domain. One critical feature is stable base‐pairing: mutations that disrupt stem pairing destabilize RNA‐OUT in vivo and abolish anti‐sense control; combinations of mutations that restore pairing also restore both stability and control. We propose that the stem renders RNA‐OUT resistant to 3′ exoribonucleases. Other features of the stem‐domain prevent this essential duplex from being an effective substrate for double‐strand nucleases: two single base mutations disrupt antisense control by making RNA‐OUT susceptible to RNase III. Mutations in the loop region have little effect on RNA‐OUT stability. Implications for IS10 biology and the design of efficient anti‐sense RNAs are discussed.

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Transcription of the replication control region of the IncFII R-plasmid NR1 in Vitro and in Vivo

Cited by 37 publications

References 39 publications

Suppression of replication-deficient mutants of IncFII plasmid NR1 can occur by two different mechanisms that increase expression of the repA1 gene

Suppression of replication-deficient mutants of IncFII plasmid NR1 can occur by two different mechanisms that increase expression of the repA1 gene

Control of replication of plasmid R1: translation of the 7k reading frame in the RepA mRNA leader region counteracts the interaction between CopA RNA and CopT RNA.

The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain.

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