An orphan gyrB in the Mycobacterium smegmatis genome uncovered by comparative genomics.

DNA gyrase is an essential topoisomerase found in all bacteria. It is encoded by gyrB and gyrA genes. These genes are organized differently in different bacteria. Direct comparison of Mycobacterium tuberculosis and Mycobacterium smegmatis genomes reveals presence of an additional gyrB in M. smegmatis flanked by novel genes. Analysis of the amino acid sequence of GyrB from different organisms suggests that the orphan GyrB in M. smegmatis may have an important cellular role.

Axial distortion as a sensor of supercoil changes: a molecular model for the homeostatic regulation of DNA gyrase.

Negative supercoiling stimulates transcription of many genes. In contrast, transcription of the genes coding for DNA gyrase is subject to a novel mechanism of autoregulation, wherein relaxation of the template DNA stimulates their transcription. Since DNA gyrase is the sole supercoiling activity in the eubacterial cell, relaxation-stimulated transcription (RST) could reflect an autoregulatory mechanism to maintain supercoil levels within the cell. Extensive deletion and mutational analyses of Escherichia coli gyrA promoter have shown that the -10 region is essential for RST; however, a molecular model has proved to be elusive. We find a strong bend centre immediately downstream of the -10 region in the gyrA promoter. On the basis of analysis of various mutants in the -10 region, we propose a model where axial distortion acts as a sensor of topological changes in DNA. Our model is consistent with earlier data with E. coli gyrA anmd gyrB promoters. We also extrapolate the model to explain the phenomenon of RST of gyr promoters in other organisms and contrast it with promoters induced by supercoiling.

Alignment and phylogenetic analysis of type II DNA topoisomerases.

DNA topoisomerases have been evolved to solve the topological problems of DNA during replication, transcription, recombination and segregation. Discovery of several new enzymes and their characterization has necessitated this compilation. This analysis shows the distinct evolutionary relatedness of type II DNA topoisomerases. A striking feature is the absence of a contiguous stretch of about 160 amino acids in one of the subunits of prokaryotic type II enzymes, which might have important implications to their structure and function.

DNA topoisomerase I from Mycobacterium smegmatis.

DNA topoisomerase I has been purified from Mycobacterium smegmatis to near homogeneity using different column chromatographic techniques. The enzyme activity relaxes form I DNA into form IV DNA, requiring Mg2+, but not ATP or any other cofactors for its activity. Several properties of the enzyme were found to be similar to that of the prototype enzyme, Escherichia coli topoisomerase I.

Control of transcription initiation.

Mechanism of control of transcription initiation have expanded far beyond the classical operon concept. Control elements are multipartite and well separated from each other. The trans-factors bound to these sites make contacts with RNA polymerase: promoter complexes by DNA bending or looping to influence the initiation event. Activators and repressors are like two faces of the same coin and their function depends on the site of action, mode of interaction with DNA and also the nutritional status of the cell.

Transcriptional specificity after mycobacteriophage I3 infection.

Transcriptional regulation following mycobacteriophage I3 infection has been investigated. For this purpose, RNA polymerase mutants (rifr) of host bacterium, Mycobacterium smegmatis have been isolated and characterised. Phage growth in rifs and rifr cells in presence of rifampicin revealed the involvement of host RNA polymerase in phage genome transcription. This was confirmed by studies on in vivo RNA synthesis as well as by direct RNA polymerase assay after phage infection. Significant stimulation in RNA polymerase activity was seen following phage infection. The maximal levels were attained in about 60 min post infection and maintained throughout the phage development period. The stimulation of polymerase activity was most pronounced when the phage DNA was used as the template. RNA polymerases from uninfected and phage-infected Mycobacterium smegmatis have been purified to homogeniety. The enzyme purification was accomplished by a rapid procedure utilising affinity chromatography on rifampicin-Sepharose columns. Subunit structure analysis of the purified RNA polymerase from uninfected and phageinfected cells showed the presence of α, β, β' and σ subunits similar to the other prokaryotic RNA polymerases. In addition, a polypeptide of 79,000 daltons was associated with the enzyme after phage infection. The enzymes differed in their properties with respect to template specificity. Phage 13 DNA was the preferred template for the modified RNA polymerase isolated from infected cells which may account for the transcriptional switch required for phage development.