RESUMEN
CopG is a 45 amino acid residue transcriptional repressor involved in the copy number control of the streptococcal plasmid pMV158. To do so, it binds to a DNA operator that contains a 13 bp pseudosymmetric DNA element. Binding of CopG to its operator results in repression, at the transcriptional level, of its own synthesis and that of the initiator of replication protein, RepB. Biochemical experiments have shown that CopG co-operatively associates to its target DNA at low protein:DNA ratios, completely protecting four helical turns on the same face of the double helix in both directions from the inverted repeat that constitutes the CopG primary target. This has been correlated with a CopG-mediated DNA bend of about 100 degrees. Here, we show that binding of CopG to DNA fragments containing the inverted repeat just at one end led to nucleation of the protein initiating from the inverted repeat. Nucleation extended to the entire fragment, with CopG-DNA contacts occurring on the same face of the DNA helix. The protein, the prototype for a family of homologous plasmid repressors, displays a homodimeric ribbon-helix-helix arrangement. It polymerises within the unbound crystal to render a continuous right-handed protein superhelix of homodimers, around which a bound double-stranded (ds) DNA could wrap. We have solved the crystal structure of CopG in complex with a 22 bp dsDNA oligonucleotide encompassing the cognate pseudosymmetric element. In the crystal, one protein tetramer binds at one face of the DNA with two parallel beta-ribbons inserted into the major groove. The DNA is bent about 50 degrees under compression of both major and minor grooves. A continuous right-handed complex helix made up mainly by protein-protein and some protein-DNA interactions is observed. The protein-protein interactions involve regions similar to those observed in the oligomerisation of the native crystals and those employed to set up the functional tetramer. A previously solved complex structure of the protein with a 19 bp dsDNA had unveiled a left-handed helical superstructure just made up by DNA interactions.
Asunto(s)
ADN Helicasas , Oligodesoxirribonucleótidos/metabolismo , Plásmidos/genética , Proteínas/química , Proteínas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Transactivadores , Transcripción Genética/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cristalografía por Rayos X , Huella de ADN , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Streptococcus/genéticaRESUMEN
Both prokaryotes and eukaryotes respond to a decrease in temperature with the expression of a specific subset of proteins. Although a large body of information concerning cold shock-induced genes has been gathered, studies on temperature regulation have not clearly identified the key regulatory factor(s) responsible for thermosensing and signal transduction at low temperatures. Here we identified a two-component signal transduction system composed of a sensor kinase, DesK, and a response regulator, DesR, responsible for cold induction of the des gene coding for the Delta5-lipid desaturase from Bacillus subtilis. We found that DesR binds to a DNA sequence extending from position -28 to -77 relative to the start site of the temperature-regulated des gene. We show further that unsaturated fatty acids (UFAs), the products of the Delta5-desaturase, act as negative signalling molecules of des transcription. Thus, a regulatory loop composed of the DesK-DesR two-component signal transduction system and UFAs provides a novel mechanism for the control of gene expression at low temperatures.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas de Unión al ADN/fisiología , Ácido Graso Desaturasas/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Proteínas de Neoplasias , Proteínas Nucleares/fisiología , Proteínas Quinasas/genética , Transducción de Señal/fisiología , Fusión Artificial Génica , Bacillus subtilis/genética , Bacillus subtilis/fisiología , Secuencia de Bases , Frío , ADN Bacteriano/metabolismo , Ácidos Grasos Insaturados/metabolismo , Genes Bacterianos , Histidina Quinasa , Operón Lac , Datos de Secuencia Molecular , Mutagénesis , Operón , Regiones Promotoras Genéticas , Proteínas Quinasas/metabolismo , ARN Bacteriano/biosíntesis , ARN Mensajero/biosíntesis , Proteínas Represoras , Factores de Transcripción , Activación Transcripcional , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Plasmid rolling circle replication generates single-stranded DNA intermediates. The intracellular amount of these molecules depends upon the efficiency of the conversion of single-stranded into double-stranded plasmid forms, that is, the functionality of the lagging strand origin (sso). The broad-host-range streptococcal plasmid pMV158 harbors two different ssos, both of which function efficiently in Streptococcus pneumoniae but poorly in Escherichia coli. Plasmid pMV158 is stably inherited in the pneumococcal host, but it is unstable in E. coli. A pMV158 derivative lacking its two ssos is unstable in both strains. We have cloned into this derivative the coliphage f1 lagging strand origin. Whereas the f1 sso was fully functional in E. coli, it did not show any activity in S. pneumoniae, a bacteria closely related to the pMV158 natural host. The presence of the f1 sso did not stabilize pMV158 inheritance in either the gram-positive or the gram-negative host.
Asunto(s)
Escherichia coli/genética , Plásmidos/genética , Clonación Molecular , Colifagos/genética , Replicación del ADN/genética , ADN de Cadena Simple/genética , Prueba de Complementación Genética , Recombinación Genética , Streptococcus pneumoniae/genéticaRESUMEN
The streptococcal plasmid pMV158 has been reported to harbor five genes: three involved in initiation of rolling circle replication and its control (copG, repB, and maII), one involved in conjugative mobilization (mobM), and the fifth one specifying constitutive resistance to tetracycline (tet). The mobM gene was removed in the construction of the pMV158-derivative plasmid pLS1, which was used in this study. By in vitro transcription assays, primer extension experiments, and construction of mutations, here we demonstrate the presence of another gene (the sixth of pMV158), termed maI, which is transcribed in opposite orientation with respect to the plasmid mRNAs, to render RNA I. The 5'-end of RNA I has an 8-nt sequence which is complementary to a region of the lagging-strand origin (ssoA) comprising a 6-nt consensus sequence involved in lagging strand synthesis. This suggested that RNA I could influence, positively or negatively, initiation of lagging strand synthesis from the pLS1-ssoA. However, plasmids defective in RNA I synthesis exhibited a phenotype similar to the wild type in terms of efficiency of replication from the ssoA and copy number. When the maI gene was cloned into a compatible plasmid, the resulting recombinants did not exhibit incompatibility toward plasmids with the pLS1 replicon. Thus, RNA I does not seem to be a true copy number control element. We postulate that transcription from the maI promoter may facilitate extrusion of the hairpin of the plasmid double-strand origin, which is the target of the initiator of replication protein.