Summary
Expression of the transfer operon in the F plasmids is negatively regulated by FinOP which has two components, the finP and finO gene products. Mutations in either gene result in increased expression of the positive regulator of transcription, traJ, leading to derepressed levels of conjugal transfer. Five mutations in the finP gene have been previously characterised by Finnegan and Willetts (1971). Three were complementable in trans and were named finP mutations and two were complementable at low levels (fisO) presumably because they affected the site of action of the finO gene product. In this study, DNA sequence analysis revealed three different mutations shared by the five mutants which were located in the stems of the predicted stem-and-loop structures in the finP anti-sense RNA. The properties of three mutants created by site-specific mutagenesis suggested that the stability of the stem structure was important in FinP action and that a small region in one of the stems appears to be the target of the finO gene product. Analysis of wild-type and fisO FinP RNA showed that FinO increased the amount of an 80 nucleotide FinP RNA, probably by stabilizing this transcript or preventing its degradation. The fisO mutation decreased the amount of 80 nucleotide RNA substantially. FinP transcripts from either the finP promoter of the lac promoter appeared to be stabilized by FinO.
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References
Achtman M, Willetts NS, Clark AJ (1971) Beginning a genetic analysis of conjugational transfer determined by the F factor in Escherichia coli by isolation and characterization of transferdeficient mutants. J Bacteriol 106:529–538
Altman S, Bowman EJ, Garber RL, Kole R, Koski RA, Stark BC (1980) Aspects of RNase P structure and function. In: Soll D, Abelson JN, Schimmel PR (eds) Transfer RNA: Biological aspects. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 71–82
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Cheah K-C, Skurray R (1986) The F plasmid carries an IS3 insertion within finO. J Gen Microbiol 132:3269–3275
Davis LG, Dibner MD, Battey JF (1986) Basis methods in molecular biology, Elsever Science Publishing Co, New York, pp 143–145
Dempsey WB (1987) Transcript analysis of the plasmid R100 traJ and finP genes. Mol Gen Genet 209:533–544
Finlay BB, Frost LS, Paranchych W, Willetts NS (1986) Nucleotide sequences of five IncF plasmid finP alleles. J Bacteriol 167:754–757
Finnegan DJ, Willetts NS (1971) Two calsses of Flac mutants insensitive to transfer inhibition by an F-like R factor. Mol Gen Genet 111:256–264
Finnegan DJ, Willetts NS (1972) The nature of the transfer inhibitior of several F-like plasmids. Mol Gen Genet 119:57–66
Finnegan DJ, Willetts NS (1973) The site of action of the F transfer inhibitor. Mol Gen Genet 127:307–316
Green PJ, Pines O, Inouye M (1986) The role of anti-sense RNA in gene regulation. Annu Rev Biochem 55:569–597
Guerrier-Takada, C, Gardiner K, Marsh T, Pace NR, Altman S (1983) The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35:849–857
Guthrie C, Atchison R (1980) Biochemical characterization of RNase P: a tRNA processing activity with protein and RNA components. In: Soll D, Abelson JN, Schimmel PR (eds) Transfer RNA: Biological aspects. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York, pp 83–97
Humphreys G, Willshaw GA, Anderson ES (1975) A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta 383:457–463
Inouye M, Delihas N (1988) Small RNAs in the prokaryotes: a growing list of diverse roles. Cell 53:5–7
Ippen-Ihler K, Minkley EG (1986) The conjugation system of F, the fertility factor of Escherichia coli. Annu Rev Genet 20:593–624
Maniatis T, Fritsch EF, Sambrook K (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
McIntire S, Dempsey WB (1987) Fertility inhibition gene of plasmid R100. Nucleic Acids Res 15:2029–2042
Meynell E, Meynell GG, Datta N (1968) Phylogenetic relationships of drug-resistance factors and other transmissible bacterial plasmids. Bacteriol Rev 32:55–83
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 328–330
Morinaga Y, Franceschini T, Inouye S, Inouye M (1984) Improvement of oligonucleotide-directed site-specific mutagenesis using double-stranded DNA. Bio/Technology 2:636–639
Mullineaux P, Willetts NS (1985) Promoters in the transfer region of plasmid F. In: Helsinki DR, Cohen SN, Clewell DB, Jackson DA, Hollaeder A (eds) Plasmids in bacteria. Plenum Publishing Corp, New York, pp 605–614
Paranchych W, Finlay BB, Frost LS (1986) Studies on the regulation of IncF plasmid transfer operon expression. In: Levy SB, Novick RP (eds) Antibiotic resistance genes: ecology, transfer, and expression. Banbury Report 24: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 117–129
Thompson R, Taylor L (1982) Promoter mapping and DNA sequencing of the F plasmid transfer genes traM and traJ. Mol Gen Genet 188:513–518
Tomizawa J, Som T (1984) Control of ColE1 plasmid replication: enhancement of binding of RNAI to the primer transcript by the Rom protein. Cell 38:871–878
Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
Wallace RB, Johnson MJ, Suggs SV, Miyoshi K, Bhatt R, Itakura K (1981) A set of synthetic oligodeoxyribonucleotide primers for DNA sequencing in the plasmids vector pBR322. Gene 16:21–26
Willetts NS, Maule J (1986) Specificities of IncF plasmid conjugation genes. Genet Res 47:1–11
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
Yoshioka Y, Ohtsubo H, Ohtsubo E (1987) Repressor gene finO in plasmids R100 and F: Constitutive transfer of plasmid F is caused by insertion of IS3 into F finO. J Bacteriol 169:619–623
Zoller MJ, Smith M (1982) Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res 10:6487–6500
Zuker M, Stiegler P (1981) Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res 9:133–148
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Communicated by N.D.F. Grindley
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Frost, L., Lee, S., Yanchar, N. et al. finP and fisO mutations in FinP anti-sense RNA suggest a model for FinOP action in the repression of bacterial conjugation by the Flac plasmid JCFLO. Mol Gen Genet 218, 152–160 (1989). https://doi.org/10.1007/BF00330578
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DOI: https://doi.org/10.1007/BF00330578