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Response of RNA polymerase to ppGpp: requirement for the omega subunit and relief of this requirement by DksA.

ABSTRACT: Previous studies have come to conflicting conclusions about the requirement for the omega subunit of RNA polymerase in bacterial transcription regulation. We demonstrate here that purified RNAP lacking omega does not respond in vitro to the effector of the stringent response, ppGpp. DksA, a transcription factor that works in concert with ppGpp to regulate rRNA expression in vivo and in vitro, fully rescues the ppGpp-unresponsiveness of RNAP lacking omega, likely explaining why strains lacking omega display a stringent response in vivo. These results demonstrate that omega plays a role in RNAP function (in addition to its previously reported role in RNAP assembly) and highlight the importance of inclusion of omega in RNAP purification protocols. Furthermore, these results suggest that either one or both of two short segments in the beta' subunit that physically link omega to the ppGpp-binding region of the enzyme may play crucial roles in ppGpp and DksA function.

SUBMITTER: Vrentas CE

PROVIDER: S-EPMC1240046 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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Response of RNA polymerase to ppGpp: requirement for the omega subunit and relief of this requirement by DksA.

Vrentas Catherine E CE Gaal Tamas T Ross Wilma W Ebright Richard H RH Gourse Richard L RL

Genes & development 20051001 19

Previous studies have come to conflicting conclusions about the requirement for the omega subunit of RNA polymerase in bacterial transcription regulation. We demonstrate here that purified RNAP lacking omega does not respond in vitro to the effector of the stringent response, ppGpp. DksA, a transcription factor that works in concert with ppGpp to regulate rRNA expression in vivo and in vitro, fully rescues the ppGpp-unresponsiveness of RNAP lacking omega, likely explaining why strains lacking om ...[more]

PMID: 16204187

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Project description:The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (?2??'? ?) containing the smallest subunit, ?. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, ?', and its recruitment in the enzyme assembly. Despite evolutionary conservation of ? and its role in the assembly of RNAP, E. coli mutants lacking rpoZ (codes for ?) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ? during transcription, several dominant lethal mutants of ? were isolated. The mutants showed higher binding affinity compared to that of native ? to the ?2??' subassembly. We observed that the interaction between ?2??' and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in rpoZ. We observed that the dominant lethal mutants of ? having either point mutation or silent in nature are more structured in comparison to the native ?. However, the silent code's position in the reading frame of rpoZ plays a role in the structural alteration of the translated protein. This structural alteration in ? makes it more rigid, which affects the plasticity of the interacting domain formed by ? and ?2??'. Here, we attempted to describe how the conformational flexibility of the ? helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ? has a suppressor mapped near the catalytic center of the ?' subunit, and it is the same for both types of mutants.

Project description:The catalytic core of the RNA polymerase of most eubacteria is composed of two M-NM-1 subunits and M-NM-2, M-NM-2M-bM-^@M-^Y and M-OM-^I subunits. In Escherichia coli, the M-OM-^I subunit (encoded by the rpoZ gene) has been suggested to assist M-NM-2M-bM-^@M-^Y during RNA polymerase core assembly. The function of the M-OM-^I subunit is particularly interesting in cyanobacteria because the cyanobacterial M-NM-2M-bM-^@M-^Y is split to N-terminal M-NM-3 and C-terminal M-NM-2M-bM-^@M-^Y subunits. The M-bM-^HM-^FrpoZ strain of the cyanobacterium Synechocystis sp. PCC 6803 grew well in standard conditions although the mutant cells showed low light-saturated photosynthetic activity, low Rubisco content and accumulated high quantities of protective carotenoids and M-NM-1-tocopherol. The M-bM-^HM-^FrpoZ strain contained 15% less of the primary M-OM-^C factor, SigA, than the control strain, and recruitment of SigA to the RNA polymerase core was inefficient in M-bM-^HM-^FrpoZ. Thus, a cyanobacterial RNA polymerase holoenzyme lacking the M-OM-^I subunit contains less frequently the primary M-OM-^C factor. A DNA microarray analysis revealed that this leads to specific down-regulation of highly expressed genes, like genes encoding subunits for Rubisco, ATP synthase, NADH-dehydrogenase and carbon concentrating mechanisms. On the contrary, many genes showing only low or moderate expression in the control strain were up-regulated in M-bM-^HM-^FrpoZ. A conserved -10 region was detected in promoters showing up or down-regulation in M-bM-^HM-^FrpoZ, but -35 regions of down-regulated genes completely differed from -35 regions of up-regulated genes. Cells from cyanobacteria Synechocystis sp. PCC 6803 named as control strain (CS) and RNA polymerase omega subunit inactivation strain, M-NM-^TrpoZ, were harvested (A730=1, 40 mL) directly from standard growth conditions (continuous illumination at the PPFD of 40 M-BM-5mol m-2s-1, 32M-BM-0C, ambient CO2). From three to four independent experiments were performed at each conditions.

Dataset Information

Response of RNA polymerase to ppGpp: requirement for the omega subunit and relief of this requirement by DksA.

Publications

Response of RNA polymerase to ppGpp: requirement for the omega subunit and relief of this requirement by DksA.

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