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Role of the coiled-coil tip of Escherichia coli DksA in promoter control.

ABSTRACT: Escherichia coli DksA works in conjunction with the small-molecule ppGpp to regulate transcription initiation negatively or positively, depending on the identity of the promoter. DksA is in a class of transcription factors that do not bind directly to DNA such as classical repressors or activators but rather bind in the RNA polymerase (RNAP) secondary channel such as the transcription elongation factors GreA and GreB in E. coli and TFIIS in eukaryotes. We found that substitution for either of two residues in its coiled-coil tip, D74 or A76, eliminates DksA function without affecting its apparent affinity for RNAP. The properties of DksA-Gre factor chimeras indicated that the coiled-coil tip is responsible for the DksA-specific effects on open complex formation. A conservative substitution at position 74, D74E, resulted in a loss of DksA function in both negative and positive control, and an E44D substitution at the analogous position in GreA resulted in a gain of function in both negative and positive control. That a single methylene group has such an extraordinary effect on these transcription factors highlights the critical nature of the identity of coiled-coil tip interactions with RNAP for open complex formation.

SUBMITTER: Lee JH

PROVIDER: S-EPMC3288215 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Role of the coiled-coil tip of Escherichia coli DksA in promoter control.

Lee Jeong-Hyun JH Lennon Christopher W CW Ross Wilma W Gourse Richard L RL

Journal of molecular biology 20111219 4

Escherichia coli DksA works in conjunction with the small-molecule ppGpp to regulate transcription initiation negatively or positively, depending on the identity of the promoter. DksA is in a class of transcription factors that do not bind directly to DNA such as classical repressors or activators but rather bind in the RNA polymerase (RNAP) secondary channel such as the transcription elongation factors GreA and GreB in E. coli and TFIIS in eukaryotes. We found that substitution for either of tw ...[more]

PMID: 22200485

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Project description:Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking of occlusion-associated proteins. IMPORTANCE:Baculovirus infection produces two forms of viruses: BV and ODV. Manufacturing of ODV involves trafficking of envelope proteins to the inner nuclear membrane, mediated partly through the FP25K protein. Since FP25K is present in alpha-, beta-, and gammabaculoviruses, it is uncertain if this trafficking function is conserved. In this study, we looked at alpha- and betabaculovirus FP25K trafficking by its localization. Alphabaculovirus FP25K localized primarily to the cytoplasm, whereas betabaculovirus FP25K localized to the nucleus. We found that an N-terminal coiled-coil domain present in all alphabaculovirus FP25K proteins, but absent in betabaculovirus FP25K, was critical for alphabaculovirus FP25K cytoplasmic localization. We believe that this represents an evolutionary process that partly led to the gain of function of this N-terminal coiled-coil domain in alphabaculovirus FP25K to aid in nuclear trafficking of occlusion-associated proteins. Due to betabaculovirus breakdown of the nuclear membrane before occlusion, this function is not needed, and the domain was lost or never acquired.

Project description:ABSTRACT DksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the "stringent response" to nutrient starvation in the gammaproteobacterium Escherichia coli and for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly related Rhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length to E. coli DksA but lacks the Zn finger motif of the E. coli DksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of an E. coli strain lacking the dksA gene and modulates transcription in vitro with E. coli RNA polymerase (RNAP) similarly to E. coli DksA. RSP2654 reduces RNAP-promoter complex stability in vitro with RNAPs from E. coli or R. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity to E. coli DksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsp has distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp. IMPORTANCE The role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis in Rhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

Dataset Information

Role of the coiled-coil tip of Escherichia coli DksA in promoter control.

Publications

Role of the coiled-coil tip of Escherichia coli DksA in promoter control.

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