Copy number control of the streptococcal plasmio plP501 occurs at three levels

Brantl, Sabine; Behnke, Detlev

doi:10.1093/nar/20.3.395

Cited by 53 publications

(70 citation statements)

References 37 publications

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“…The plasmids used in this study to construct new derivatives of pIP501 have been described previously (9,10). Plasmids were propagated in Bacillus subtilis DB104 (24), and Eschenichia coli TG2 (35) was used for subcloning and mutagenesis experiments.…”

Section: Materlils and Methodsmentioning

confidence: 99%

“…The copy number of all pIP501 derivatives in B. subtilis DB104 was determined as described before (9). Mutations within RNAIII affect the copy number.…”

Section: Materlils and Methodsmentioning

confidence: 99%

“…One level involves the inverted repeat upstream of promoter p1I, which is likely to be the target of a negative autoregulation by RepR. There is striking similarity of sequences within IR1 and the origin region (7,9). The second level includes the CopR protein and the antisense RNAIII.…”

mentioning

confidence: 99%

“…The second level includes the CopR protein and the antisense RNAIII. Mutations in either regulatory component cause the same copy effect but are not additive, and mutations within RNAIII or its promoter pIII can be detected only in the presence of CopR (9). The antisense RNAIII can assume a stable secondary structure, which suggested a functional importance of certain single-stranded loops.…”

mentioning

confidence: 99%

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RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism

Brantl¹,

Birch-Hirschfeld²,

Behnke³

1993

J Bacteriol

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Expression of the rate-limiting initiator protein RepR of plasmid pIP501 is controlled by the antisense RNAIII. Mutational alteration of individual G residues within the single-stranded loops of RNAIII led to an increase in copy number. In contrast to the G-rich single-stranded loops, two smaller AT-rich loops of RNAIII were found to be dispensable for its inhibitory function. Reciprocal mutations in the same loop compensated for each other's effect, and a destabilization of the major stem structure of RNAIII also resulted in an increased copy number. These data were consistent with the idea that the interaction of RNAIII with its target starts with the formation of a kissing complex between the single-stranded loops of both molecules. The repR mRNA leader sequence, which includes the target of RNAIIIs is able to assume two alternative structures due to the presence of two inverted repeats the individual sequences of which are mutually complementary. In the presence of the antisense RNAIII, one of these inverted repeats (IR2) is forced to fold into a transcriptional terminator structure that prevents transcription of the repR gene. In the absence of RNAIII, formation of the transcriptional terminator is prevented and expression of the essential repR gene can proceed normally. This antisense RNA-driven transcriptional attenuation mechanism was supported by extensive deletional analysis and direct evidence that IR2 functions as a transcriptional terminator.

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Section: Materlils and Methodsmentioning

confidence: 99%

“…The copy number of all pIP501 derivatives in B. subtilis DB104 was determined as described before (9). Mutations within RNAIII affect the copy number.…”

Section: Materlils and Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism

Brantl¹,

Birch-Hirschfeld²,

Behnke³

1993

J Bacteriol

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“…However, this structure is not required for replication, which sets pAMp1 apart from the previously described Repdependent class A plasmids. It is conceivable that this structure plays a role in regulation of plasmid copy number, since it is located in the vicinity of signals involved in the control ofORF E transcription (42,43) replication. Two types of models can be considered.…”

Section: Discussionmentioning

confidence: 99%

A fourth class of theta-replicating plasmids: the pAM beta 1 family from gram-positive bacteria.

Bruand

Chatelier

Ehrlich

et al. 1993

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

110

128

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Plasmid pAMfil from Enterococcus faecalis uses a unidirectional theta mode of replication. We show here that this replication (i) is dependent on a plasmid-encoded replication protein (Rep) but not on a DNA structure typical for origins of most Rep-dependent plasmids and (ii) is initiated by DNA polymerase I (PolI). pAMI31 minimal replicon shares no homology with highly conserved ColEl-type replicons, which use Poll for initiation but do not encode a Rep, or with CoIE2 and CoIE3 replicons, which require Poll for replication and encode a Rep. We propose that pAM,B1 and a number of other naturally occurring and closely related plasmids form a distinct plasmid class.Circular bacterial plasmids use two modes of DNA replication, known as rolling circle and theta. The former, first found for single-stranded DNA phages of Escherichia coli, is frequently used by small (< 10-kb) plasmids from Gram-positive bacteria (ref.

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Transcriptional repressor CopR: Structure model-based localization of the deoxyribonucleic acid binding motif

Steinmetzer

Hillisch²,

Behlke

et al. 2000

Proteins

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The plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number. CopR binds as a dimer to a nearly palindromic operator with the consensus sequence 5'-CGTG. Intermediate sequence searches revealed a significant structural relationship between CopR and the bacteriophage P22 c2 and the 434 c1 repressors. In this report we describe the experimental verification of a CopR homology model, which is based on a fairly low-sequence identity of 13.8% to P22 c2 repressor. A model for the complex of CopR with the deoxyribonucleic acid (DNA) target was built on the basis of experimental footprinting data, the above-mentioned CopR homology model, and the crystal structure of the 434 c1 repressor-DNA complex. Site-directed mutagenesis was used to test the function of amino acids involved in sequence and nonsequence-specific DNA recognition and amino acids important for correct protein folding. CD measurements were performed to detect structural changes caused by the mutations. Exchanges of residues responsible for sequence-specific DNA recognition reduced binding to a nonspecific level. Mutations of amino acids involved in nonspecific DNA binding lead to decreased binding affinity while maintaining selectivity. Substitution of amino acids necessary for proper folding caused dramatic structural changes. The experimental data support the model of CopR as a helix-turn-helix protein belonging to the lambda repressor superfamily.

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Copy number control of the streptococcal plasmio plP501 occurs at three levels

Cited by 53 publications

References 37 publications

RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism

RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism

A fourth class of theta-replicating plasmids: the pAM beta 1 family from gram-positive bacteria.

Transcriptional repressor CopR: Structure model-based localization of the deoxyribonucleic acid binding motif

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