Fernando Rojo

Bend induced by the phage φ29 transcriptional activator in the viral late promoter is required for activation

Transcription initiation from the Bacillus subtilis phage phi 29 late A3 promoter requires the viral protein p4, a transcriptional activator. Protein p4 binds to a region of the A3 promoter, located between nucleotides -50 and -100 relative to the transcription start site, that presents a sequence-directed curvature. This curvature is enhanced when protein p4 binds to the promoter. A number of deletion mutants at the carboxyl end of protein p4 have been constructed and their behavior as transcriptional activators of the late A3 promoter has been investigated. The binding of these deletion mutants to the late A3 promoter has been analyzed by gel retardation, DNase I footprinting, methylation interference and circular permutation assays. The results suggest that the last 12 amino acid residues of protein p4, six of which are positively charged, although not involved in the specific recognition of the promoter are responsible for part of the bend induced by protein p4 in its binding site. Evidence is presented which suggests that full induction of this curvature is needed for the transcription activation process. A model is proposed for protein p4 interaction with the A3 promoter, in which the bend is induced in two steps: first, two monomers of protein p4 bind to the inverted recognition sequences, subsequent interaction between them generating a bend between these sequences; second, the highly basic carboxyl terminus of protein p4 establishes non-specific electrostatic interactions with the DNA backbone inducing a bend at both ends of the protein p4 binding region.

Catabolite Repression and Physiological Control

When confronted with a mixture of potential carbon sources at sufficiently high concentrations, many bacterial species often assimilate the different compounds in an ordered fashion, so that expression of the pathway for the assimilation of the non-preferred substrate remains inhibited until the preferred one is consumed. Pseudomonads are no exception to this. This phenomenon is, at least at first sight, similar to the “catabolite repression” control initially described in Escherichia coli for the hierarchi cal assimilation of sugars39. Catabolite repression has been studied mainly in E. itcoli and Bacillus subtilis, where it was found to be the consequence of a complex global regulatory response. In E. coli, the transport of glucose by the phosphotransferase sugar transport system (PTS) is coupled to its phosphorylation, a process that activates mechanisms to impede the import of other sugars32, 55. This process, named inducer exclusion, prevents expression of the catabolic pathways for other alternative sugars by lowering the intracellular concentration of the corresponding inducer. When glucose is consumed the levels of cAMP increase to levels that allow its binding to the cAMP receptor protein (CRP). The cAMP-CRP protein can then bind to a large number of promoters, where it acts as a transcriptional activator or repressor32,55. Many of the activated promoters correspond to catabolic pathways for other alternative sugars. A protein called Cra also regulates the metabolism of carbohydrates in E. coli.

The Bacillus subtilis chromatin‐associated protein Hbsu is involved in DNA repair and recombination

The Bacillus subtilis hbs gene encodes an essential chromatin‐associated protein termed Hbsu. Hbsu, the counterpart of the Escherichia coli HU protein, binds DNA in a non‐specific way but has a clear preference for bent, kinked or altered DNA sequences. To investigate the role of Hbsu in DNA repair and DNA recombination we have constructed a series of site‐directed mutants in the hbs gene and used these mutant genes to substitute the wild‐type chromosomal hbs gene. The hbs47 mutation, which codes for a mutant protein in which residue Phe‐47 has been replaced by Trp, does not cause any discernible phenotype. Additional substitution of residue Arg‐55 by Ala (hbs4755 mutation) rendered cells deficient in DNA repair, homologous recombination and (i protein‐mediated site‐specific recombination. We have also tested the effect on DNA repair of the hbs4755 mutation in combination with mutations in different functions of homologous DNA recombination (recA, recF, recG, recti and addAB). The hbs4755 mutation did not modify the sensitivity of recH and addAB cells to the DNA‐damaging agents methylmethane sulphonate (MMS) or 4‐nitroquinoline‐1‐oxide (4NQO), and it only marginally affected recF and recG cells. The hbs4755 mutation blocked intermolecular recombination in recH cells and markedly reduced it (20‐ to 50‐fold) in recF and recG cells, but had no effect on addAB cells. Taken together, these data indicate that the Hbsu protein is required for DNA repair and for homologous DNA recombination.

Short N-terminal deletions in the phage φ29 transcriptional activator protein impair its DNA-binding ability

The expression of Bacillus subtilis phage phi 29 late genes from the A3 promoter requires the viral protein p4. This protein is a transcriptional activator which binds to a region of the A3 promoter located between nucleotides -56 to -102, relative to the transcription start point. Mutants at the N terminus of protein p4 have been constructed and their function investigated. The binding of these deletion mutants to the late A3 promoter has been analyzed by gel retardation and DNase I footprinting assays. The results indicate that the N terminus of protein p4 could be involved in its binding to the A3 promoter, suggesting that it may not be a typical Cro-like helix-turn-helix DNA-binding protein.