Nucleic Acids Research, Vol 25, Issue 19 3840-3846, Copyright © 1997 by Oxford University Press
P Stolt and NG Stoker
The regulatory region of the Mycobacterium fortuitum plasmid pAL5000 was
studied in vivo and in vitro by mutational analysis. This region comprises
the origin of replication for the plasmid and the start point of
transcription for the repA/B genes, which encode the two replication
proteins RepA and RepB. In this region there are two binding sites for
RepB: a low-affinity site which is probably the origin of replication and a
high-affinity-site which overlaps the promoter and implies an autoregulated
expression of RepB. The high-affinity site contains two 8 bp palindromes,
as well as an inverted repeat structure. By introducing point mutations
into each of these motifs and monitoring changes to RepB binding in a
gel-retardation assay, it was shown that the central, GC-rich palindrome
(the GC-box) is the most important motif for protein binding. Mutations in
the second, AT-rich palindrome (the AT-box) had no effect on protein
binding and the inverted repeat structure per se was not needed, though
some single-base changes affected binding to one or other of the DNA
strands. These mutations were subsequently tested in vivo for their effects
on plasmid replication in Mycobacterium smegmatis. Any change to the GC-box
abolished replication, but changes to the other motifs were dependent on
the position of the changed base, again indicating that the inverted
repeats are not essential and that the AT-box is part of the promoter and
not primarily recognised by RepB. The mutated plasmids did not show any
changes in copy number to that of the wild-type. The expression of RepB was
boosted by introducing a stronger promoter upstream of the repA/B genes.
The resulting plasmid was capable of increasing to a degree in trans the
copy number of other plasmids carrying the ori region, but was unstable
when present on its own in M.smegmatis.
ARTICLES
Mutational analysis of the regulatory region of the Mycobacterium plasmid pAL5000
Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK. pstolt@fz-borstel.de
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Chatterjee, A. Basu, A. Basu, and S. K. Das Gupta DNA Bending in the Mycobacterial Plasmid pAL5000 Origin-RepB Complex J. Bacteriol., December 1, 2007; 189(23): 8584 - 8592. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Mo, N. M. Quanquin, W. H. Vecino, U. D. Ranganathan, L. Tesfa, W. Bourn, K. M. Derbyshire, N. L. Letvin, W. R. Jacobs Jr., and G. J. Fennelly Genetic Alteration of Mycobacterium smegmatis To Improve Mycobacterium-Mediated Transfer of Plasmid DNA into Mammalian Cells and DNA Immunization Infect. Immun., October 1, 2007; 75(10): 4804 - 4816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Basu, S. Chatterjee, and S. K. Das Gupta Translational Coupling to an Upstream Gene Promotes Folding of the Mycobacterial Plasmid pAL5000 Replication Protein RepB and Thereby Its Origin Binding Activity J. Bacteriol., January 15, 2004; 186(2): 335 - 342. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Basu, M. Chawla-Sarkar, S. Chakrabarti, and S. K. Das Gupta Origin Binding Activity of the Mycobacterial Plasmid pAL5000 Replication Protein RepB Is Stimulated through Interactions with Host Factors and Coupled Expression of repA J. Bacteriol., April 15, 2002; 184(8): 2204 - 2214. [Abstract] [Full Text]
|
|
|
|
 |
|
 G. Bachrach, M. J. Colston, H. Bercovier, D. Bar-Nir, C. Anderson, and K. G. Papavinasasundaram A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon Microbiology, February 1, 2000; 146(2): 297 - 303. [Abstract] [Full Text]
|
|