An Antisense RNA-Mediated Transcriptional Attenuation Mechanism Functions in Escherichia coli

ABSTRACT Antisense RNA-mediated transcriptional attenuation is a regulatory mechanism operating in the replication control of two groups of plasmids in gram-positive bacteria, the pT181 group and the inc18 family, represented by pIP501. In contrast, this control mechanism has so far not been identified in gram-negative bacteria or their plasmids. In this work we asked whether such a mechanism can be supported by Escherichia coli. The core replication control regions of plasmids pT181 and pIP501 were transferred into this heterologous host. In vivo lacZ reporter gene assays showed that the antisense RNAs of these plasmids can inhibit lacZ expression and that most of this effect can be accounted for by reduced mRNA readthrough. Northern analyses confirmed that the ratio of attenuated to readthrough target RNA was increased in the presence of the cognate antisense RNA, as expected for this mechanism. Similarly, both antisense RNAs induced premature termination of their cognate target RNAs in an E. coli in vitro transcription system, whereas the noncognate antisense RNAs had no effect. Thus, this report shows that antisense RNA-mediated transcriptional attenuation is supported by at least one gram-negative host, although the data indicate that inhibitory efficiencies are lower than those for, e.g., Bacillus subtilis. Possible explanations for the apparent absence of this control mode in plasmids of gram-negative bacteria are discussed.

[1]  S. Brantl,et al.  Copy number control of the streptococcal plasmid pIP501 occurs at three levels. , 1992, Nucleic Acids Research.

[2]  E. Wagner,et al.  Kinetic aspects of control of plasmid replication by antisense RNA. , 1994, Trends in biochemical sciences.

[3]  R. Burgess,et al.  RNA Polymerases from Bacillus subtilisand Escherichia coli Differ in Recognition of Regulatory Signals In Vitro , 2000, Journal of bacteriology.

[4]  J. Kornblum,et al.  pT181 plasmid replication is regulated by a countertranscript-driven transcriptional attenuator , 1989, Cell.

[5]  E. Wagner,et al.  An unusually long-lived antisense RNA in plasmid copy number control: in vivo RNAs encoded by the streptococcal plasmid pIP501. , 1996, Journal of molecular biology.

[6]  H. Margalit,et al.  Novel small RNA-encoding genes in the intergenic regions of Escherichia coli , 2001, Current Biology.

[7]  A. Nicholson.  Function, mechanism and regulation of bacterial ribonucleases. , 1999, FEMS microbiology reviews.

[8]  E. Wagner,et al.  Kissing and RNA stability in antisense control of plasmid replication. , 1998, Trends in biochemical sciences.

[9]  S. Brantl.  The copR gene product of plasmid p1P501 acts as a transcriptional repressor at the essential repR promoter , 1994, Molecular microbiology.

[10]  E. Chatelier,et al.  Countertranscript‐driven attenuation system of the pAMβ1 repE gene , 1996, Molecular microbiology.

[11]  J. Alonso,et al.  Molecular analysis of the replication region of the conjugative Streptococcus agalactiae plasmid pIP501 in Bacillus subtilis. Comparison with plasmids pAM beta 1 and pSM19035. , 1990, Nucleic acids research.

[12]  Antisense RNA. , 1990, Critical reviews in biochemistry and molecular biology.

[13]  C. J. McGrath,et al.  Effect of exchange rate return on volatility spill-over across trading regions , 2012 .

[14]  G. Venema,et al.  Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli , 1984, Applied and environmental microbiology.

[15]  G. Storz,et al.  Identification of novel small RNAs using comparative genomics and microarrays. , 2001, Genes & development.

[16]  K. Nordström,et al.  A runaway-replication mutant of plasmid R1drd-19: Temperature-dependent loss of copy number control , 1978, Molecular and General Genetics MGG.

[17]  E. Wagner,et al.  Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III. , 1990, The EMBO journal.

[18]  E. Wagner,et al.  Dual function of the copR gene product of plasmid pIP501 , 1997, Journal of bacteriology.

[19]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[20]  S. Brantl,et al.  In vitro and in vivo analysis of transcription within the replication region of plasmid pIP501 , 1992, Molecular and General Genetics MGG.

[21]  R. Simons,et al.  Antisense RNA control in bacteria, phages, and plasmids. , 1994, Annual review of microbiology.

[22]  M. Ehrenberg,et al.  Molecular clocks reduce plasmid loss rates: the R1 case. , 2000, Journal of molecular biology.

[23]  E. Wagner,et al.  An antisense/target RNA duplex or a strong intramolecular RNA structure 5' of a translation initiation signal blocks ribosome binding: the case of plasmid R1. , 1996, RNA.

[24]  S. Brantl,et al.  RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism , 1993, Journal of bacteriology.

[25]  Siddhartha Roy,et al.  Activation and Repression of Transcription by Differential Contact: Two Sides of a Coin* , 1998, The Journal of Biological Chemistry.

[26]  C. Kumar,et al.  Plasmid pT181 replication is regulated by two countertranscripts. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[27]  T. Henkin.  Control of transcription termination in prokaryotes. , 1996, Annual review of genetics.

[28]  Jeffrey W. Roberts,et al.  Mechanism of intrinsic transcription termination and antitermination. , 1999, Science.

[29]  E. Wagner,et al.  Antisense RNAs in bacteria and their genetic elements. , 2002, Advances in genetics.

[30]  C. Yanofsky.  Transcription Attenuation: Once Viewed as a Novel Regulatory Strategy , 2000, Journal of bacteriology.

[31]  E. Wagner,et al.  Antisense RNA‐mediated transcriptional attenuation occurs faster than stable antisense/target RNA pairing: an in vitro study of plasmid pIP501. , 1994, The EMBO journal.

[32]  S. Brantl,et al.  The amount of RepR protein determines the copy number of plasmid pIP501 in Bacillus subtilis , 1992, Journal of bacteriology.

[33]  E. Wagner,et al.  Antisense RNA‐mediated transcriptional attenuation: an in vitro study of plasmid pT181 , 2000, Molecular microbiology.

[34]  R. Novick,et al.  The effect of plasmid copy number mutations on pT181 replication initiator protein expression. , 1991, Plasmid.

[35]  G. Storz,et al.  Small RNAs in Escherichia coli. , 1999, Trends in microbiology.

[36]  M. Ehrenberg.  Hypothesis: hypersensitive plasmid copy number control for ColE1. , 1996, Biophysical journal.