RESUMO
Expression of several heat shock operons, mainly coding for small heat shock proteins, is under the control of ROSE (repression of heat shock gene expression) in various rhizobial species. This negatively cis-acting element confers temperature control by preventing expression at physiological temperatures. We provide evidence that ROSE-mediated regulation occurs at the post-transcriptional level. A detailed mutational analysis of ROSE(1)-hspA translationally fused to lacZ revealed that its highly conserved 3'-half is required for repression at normal temperatures (30 degrees C). The mRNA in this region is predicted to form an extended secondary structure that looks very similar in all 15 known ROSE elements. Nucleotides involved in base pairing are strongly conserved, whereas nucleotides in loop regions are more divergent. Base substitutions leading to derepression of the lacZ fusion at 30 degrees C exclusively resided in potential stem structures. Optimised base pairing by elimination of a bulged residue and by introduction of complementary nucleotides in internal loops resulted in ROSE elements that were tightly repressed not only at normal but also at heat shock temperatures. We propose a model in which the temperature-regulated secondary structure of ROSE mRNA influences heat shock gene expression by controlling ribosome access to the ribosome-binding site.
Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico , Processamento Pós-Transcricional do RNA , RNA Bacteriano/fisiologia , Rhizobiaceae/genética , Região 3'-Flanqueadora , Pareamento de Bases , Sequência de Bases , Análise Mutacional de DNA , Inativação Gênica , Modelos Genéticos , Conformação de Ácido Nucleico , Óperon , RNA Bacteriano/química , RNA Mensageiro/química , RNA Mensageiro/fisiologia , Sequências Reguladoras de Ácido Nucleico , Rhizobiaceae/metabolismoRESUMO
Expression of at least ten genes in Bradyrhizobium japonicum, seven of which code for small heat shock proteins (sHsps), is under the control of ROSE (repression of heat shock gene expression). This negatively cis-acting DNA element confers temperature control to a sigma(70)-type promoter. Here, we show that ROSE elements are not restricted to B. japonicum but are also present in Bradyrhizobium sp. (Parasponia), Rhizobium sp. strain NGR234 and Mesorhizobium loti. An overall alignment of all ROSE sequences reveals a highly conserved and probably functionally important region towards the 3'-end of the element. Moreover, we provide genetic evidence for the previously proposed presence of multiple sHsps in these organisms. Primer-extension data of five newly identified ROSE-associated operons show that transcription is repressed at low temperatures and induced after a temperature upshift. Translational ROSE-hsp'-'lacZ fusions of Bradyrhizobium sp. (Parasponia) and Rhizobium sp. strain NGR234 integrated into the chromosome of B. japonicum were heat-responsive. The functionality of these heterologous ROSE elements hints at a common regulatory principle conserved in various rhizobia.