scholarly journals Pseudoknot-Dependent Translational Coupling in repBA Genes of the IncB Plasmid pMU720 Involves Reinitiation

2002 ◽  
Vol 184 (20) ◽  
pp. 5772-5780 ◽  
Author(s):  
J. Praszkier ◽  
A. J. Pittard
Keyword(s):  
Antisense Rna ◽  
Copy Number ◽  
Unusual Feature ◽  
Leader Peptide ◽  
Translational Coupling ◽  
Initiator Protein ◽  
Replication Initiator Protein ◽  
Shine Dalgarno Sequence ◽  
Replication Initiator

ABSTRACT Replication of the IncB miniplasmid pMU720 requires synthesis of the replication initiator protein, RepA, whose translation is coupled to that of a leader peptide, RepB. The unusual feature of this system is that translational coupling in repBA has to be activated by the formation of a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence. A small antisense RNA, RNAI, controls replication of pMU720 by interacting with repBA mRNA to inhibit expression of repA both directly, by preventing formation of the pseudoknot, and indirectly, by inhibiting translation of repB. The mechanism of translational coupling in repBA was investigated using the specialized ribosome system, which directs a subpopulation of ribosomes that carry an altered anti-Shine-Dalgarno sequence to translate mRNA molecules whose Shine-Dalgarno sequences have been altered to be complementary to the mutant anti-Shine-Dalgarno sequence. Our data indicate that translation of repA involves reinitiation by the ribosome that has terminated translation of repB. The role of the pseudoknot in this process and its effect on the control of copy number in pMU720 are discussed.

scholarly journals Analysis of Elements Involved in Pseudoknot-Dependent Expression and Regulation of the repA Gene of an IncL/M Plasmid

1999 ◽  
Vol 181 (6) ◽  
pp. 1811-1819 ◽  
Author(s):  
V. Athanasopoulos ◽  
J. Praszkier ◽  
A. J. Pittard
Keyword(s):  
Antisense Rna ◽  
Start Codon ◽  
Leader Peptide ◽  
Initiator Protein ◽  
Repa Gene ◽  
Replication Initiator Protein ◽  
Shine Dalgarno Sequence ◽  
Rna Pseudoknot ◽  
Replication Initiator ◽  
Maximal Expression

ABSTRACT Replication of the IncL/M plasmid pMU604 is controlled by a small antisense RNA molecule (RNAI), which, by inhibiting the formation of an RNA pseudoknot, regulates translation of the replication initiator protein, RepA. Efficient translation of the repA mRNA was shown to require the translation and correct termination of the leader peptide, RepB, and the formation of the pseudoknot. Although the pseudoknot was essential for the expression of repA, its presence was shown to interfere with the translation ofrepB. The requirement for pseudoknot formation could in large part be obviated by improving the ribosome binding region ofrepA, either by replacing the GUG start codon by AUG or by increasing the spacing between the start codon and the Shine-Dalgarno sequence (SD). The spacing between the distal pseudoknot sequence and the repA SD was shown to be suboptimal for maximal expression of repA.

The effect of plasmid copy number mutations on pT181 replication initiator protein expression

Plasmid ◽  
1991 ◽  
Vol 25 (3) ◽  
pp. 198-207 ◽  
Author(s):  
Pei-Zhi Wang ◽  
Vitalia B. Henriquez ◽  
Steven J. Projan ◽  
Serban Iordanescu ◽  
Richard P. Novick
Keyword(s):  
Protein Expression ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Initiator Protein ◽  
Replication Initiator Protein ◽  
Replication Initiator

scholarly journals Role of RepA and DnaA Proteins in the Opening of the Origin of DNA Replication of an IncB Plasmid

2004 ◽  
Vol 186 (12) ◽  
pp. 3785-3793 ◽  
Author(s):  
T. Betteridge ◽  
J. Yang ◽  
A. J. Pittard ◽  
J. Praszkier
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Origin Of Replication ◽  
Initiator Protein ◽  
Origin Of Dna Replication ◽  
Replication Initiator Protein ◽  
Replication Initiator

ABSTRACT The replication initiator protein RepA of the IncB plasmid pMU720 was shown to induce localized unwinding of its cognate origin of replication in vitro. DnaA, the initiator protein of Escherichia coli, was unable to induce localized unwinding of this origin of replication on its own but enhanced the opening generated by RepA. The opened region lies immediately downstream of the last of the three binding sites for RepA (RepA boxes) and covers one turn of DNA helix. A 6-mer sequence, 5′-TCTTAA-3′, which lies within the opened region, was essential for the localized unwinding of the origin in vitro and origin activity in vivo. In addition, efficient unwinding of the origin of replication of pMU720 in vitro required the native positioning of the binding sites for the initiator proteins. Interestingly, binding of RepA to RepA box 1, which is essential for origin activity, was not required for the localized opening of the origin in vitro.

scholarly journals Replication Control of Staphylococcal Multiresistance Plasmid pSK41: an Antisense RNA Mediates Dual-Level Regulation of Rep Expression

2006 ◽  
Vol 188 (12) ◽  
pp. 4404-4412 ◽  
Author(s):  
Stephen M. Kwong ◽  
Ronald A. Skurray ◽  
Neville Firth
Keyword(s):  
Translation Initiation ◽  
Antisense Rna ◽  
Copy Number ◽  
Translation Initiation Region ◽  
Stem Loop ◽  
Initiator Protein ◽  
Stem Loop Structure ◽  
Secondary Structure Predictions ◽  
Replication Control ◽  
Replication Initiator

ABSTRACT Replication of staphylococcal multiresistance plasmid pSK41 is negatively regulated by the antisense transcript RNAI. pSK41 minireplicons bearing rnaI promoter (P rnaI ) mutations exhibited dramatic increases in copy number, approximately 40-fold higher than the copy number for the wild-type replicon. The effects of RNAI mutations on expression of the replication initiator protein (Rep) were evaluated using transcriptional and translational fusions between the rep control region and the cat reporter gene. The results suggested that when P rnaI is disrupted, the amount of rep mRNA increases and it becomes derepressed for translation. These effects were reversed when RNAI was provided in trans, demonstrating that it is responsible for significant negative regulation at two levels, with the greatest repression exerted on rep translation initiation. Mutagenesis provided no evidence for RNAI-mediated transcriptional attenuation as a basis for the observed reduction in rep message associated with expression of RNAI. However, RNA secondary-structure predictions and supporting mutagenesis data suggest a novel mechanism for RNAI-mediated repression of rep translation initiation, where RNAI binding promotes a steric transition in the rep mRNA leader to an alternative thermodynamically stable stem-loop structure that sequesters the rep translation initiation region, thereby preventing translation.

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