RESUMEN
HspR is a repressor known to control expression of heat shock operons in a number of Eubacteria. In mycobacteria and in several other actinobacteria, this protein is synthesized from the dnaKJE-hspR operon. Previous investigations revealed that HspR binds to the operon promoter, thereby controlling its expression in an autoregulatory manner. DnaK, which is a product of the same operon, further aids this autoregulatory process by stimulating the operator binding activity of HspR. The molecular mechanism by which DnaK assists HspR in executing its function is not clearly understood. In this study, it has been shown that DnaK can augment DNA binding activity of HspR by two mechanisms: (i) DnaK can restore the activity of completely denatured HspR by forming a complex with it, and (ii) DnaK can prevent thermal instability of HspR renatured by other means. Unlike the first mechanism, the latter function does not involve complex formation. The C-terminal hydrophobic tail of HspR was found to play a significant role in determining its thermal stability and DnaK dependence properties. A deletion mutant in which this region is removed does not respond to thermal stress and functions independent of DnaK. The hydrophobic C-terminal tails of HspRs of Mycobacterium tuberculosis and related Actinomycetales therefore may have evolved to make these HspRs more sensitive to thermal stress and, at the same time, subject to regulation by DnaK.
Asunto(s)
Proteínas Bacterianas/química , ADN Bacteriano/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Interacciones Hidrofóbicas e Hidrofílicas , Chaperonas Moleculares/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Filogenia , Regiones Promotoras Genéticas , Alineación de Secuencia , Eliminación de Secuencia , Transcripción GenéticaRESUMEN
In Mycobacterium tuberculosis, hspR is the last gene of the dnaKJE operon. It encodes the repressor HspR, which regulates the expression from this operon by binding to a consensus upstream sequence known as HAIR (HspR-associated inverted repeats). Previous investigations in the related Gram-positive bacterium Streptomyces coelicolor have revealed that DnaK acts as a co-repressor for HspR. In this investigation, a similar situation was encountered using the corresponding mycobacterial pair. However, the novel feature unearthed in this study is that the mycobacterial GroELs, GroEL1 and GroEL2, considerably stimulate the HAIR-binding activity of the HspR-DnaK combination. That these GroELs play a role in the folding process was evident from the observation that when heat- or chemically denatured HspR was renatured, the protein gained optimal activity only if one of these GroEL class chaperones was present along with DnaK. The renaturation process was found to be dependent on ATP hydrolysis. The DnaK-dependent DNA-binding activity of HspR could also be stimulated by DnaJ, but GrpE, which is known to release DnaK-bound substrates, was found to be inhibitory. The results of this study suggest that protein folding plays a substantial role in the activation of HspR following heat shock and that DnaK may be involved in two ways -- first, as a chaperone acting in concert with GroEL and/or DnaJ and second, as a co-repressor bound to HspR.
