Alternative sigma factors in the free state are equilibrium mixtures of open and compact conformations

Conformational switching upon core RNA polymerase binding is an integral part of functioning of bacterial sigma factors. Here, we have studied dynamical features of two alternative sigma factors. A study of fluorescence resonance energy transfer and hydrodynamic measurements in Escherichia coli σ(32) suggest a compact shape like those found in complex with anti-sigma factors. On the other hand, the fluorescence anisotropy of probes attached to different regions of the protein and previous hydrogen exchange measurements suggest significant internal flexibility, particularly in the C-terminal half and region 1. In a homologous sigma factor, σ(F) of Mycobacterium tuberculosis, emission spectra and fluorescence resonance energy transfer between the single tryptophan (W112) and probes placed in different regions suggest a compact conformation for a major part of the N-terminal half encompassing region 2 and the flexible C-terminal half. Fluorescence anisotropy measurements suggest significant flexibility in the C-terminal half and region 1, as well. Thus, free alternative sigma factors may be in equilibrium between two conformations: a compact one in which the promoter interacting motifs are trapped in the wrong conformation and another less abundant one with a more open and flexible conformation. Such flexibility may be important for promoter recognition and interaction with many partner proteins.     

The reovirus cell attachment protein possesses two independently active trimerization domains: basis of dominant negative effects.

The reovirus cell attachment protein, sigma 1, is a homotrimer with an N-terminal fibrous tail and a C-terminal globular head. By cotranslating full-length and various truncated sigma 1 proteins in vitro, we show that the N- and C-terminal halves of sigma 1 possess independent trimerization and folding domains. Trimerization of sigma 1 is initiated at the N-terminus by the formation of a "loose," protease-sensitive, three-stranded, alpha-helical coiled coil. This serves to bring the three unfolded C-termini into close proximity to one another, facilitating their subsequent trimerization and cooperative folding. Concomitant with, but independent of, this latter process, the N-terminal fiber further matures into a more stable and protease-resistant structure. The coordinated folding of sigma 1 trimers exemplifies the dominant negative effects of mutant subunits in oligomeric complexes.     

Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme

We affinity-purified the tobacco plastid-encoded plastid RNA polymerase (PEP) complex by the alpha subunit containing a C-terminal 12 x histidine tag using heparin and Ni(2+) chromatography. The composition of the complex was determined by mass spectrometry after separating the proteins of the >900 kDa complex in blue native and SDS polyacrylamide gels. The purified PEP contained the core alpha, beta, beta', beta" subunits and five major associated proteins of unknown function, but lacked sigma factors required for promoter recognition. The holoenzyme efficiently recognized a plastid psbA promoter when it was reconstituted from the purified PEP and recombinant plastid sigma factors. Reconstitution of a plastid holoenzyme with individual sigma factors will facilitate identification of sigma factor-specific promoter elements.     

Structural basis of DNA recognition by the alternative sigma-factor, sigma54

The sigma subunit of bacterial RNA polymerase (RNAP) regulates gene expression by directing RNAP to specific promoters. Unlike sigma(70)-type proteins, the alternative sigma factor, sigma(54), requires interaction with an ATPase to open DNA. We present the solution structure of the C-terminal domain of sigma(54) bound to the -24 promoter element, in which the conserved RpoN box motif inserts into the major groove of the DNA. This structure elucidates the basis for sequence specific recognition of the -24 element, orients sigma(54) on the promoter, and suggests how the C-terminal domain of sigma(54) interacts with RNAP.     

Functional interactions between the noncovalently associated N- and C-terminal halves of mammalian Type I hexokinase

The 100 kDa Type I isozyme of mammalian hexokinase has evolved by duplication and fusion of a gene encoding an ancestral 50 kDa hexokinase. Although the N- and C-terminal halves are similar in sequence, they differ in function, catalytic activity being associated only with the C-terminal half while the N-terminal half serves a regulatory role. The N- and C-terminal halves of rat Type I hexokinase have been coexpressed in M + R 42 cells. The halves associate noncovalently to produce a 100 kDa form that exhibits characteristics seen with the intact Type I isozyme but not with the isolated catalytic C-terminal half, i.e., characteristics that are influenced by interactions between the halves. These include a decreased K(m) for the substrate ATP and the ability of P(i) to antagonize inhibition by Glc-6-P or its analog, 1-5-anhydroglucitol-6-P. Thus, functional interactions between the N- and C-terminal halves do not require their covalent linkage.     

Secondary transporters of the 2HCT family contain two homologous domains with inverted membrane topology and trans re-entrant loops

The 2-hydroxycarboxylate transporter (2HCT) family of secondary transporters belongs to a much larger structural class of secondary transporters termed ST3 which contains about 2000 transporters in 32 families. The transporters of the 2HCT family are among the best studied in the class. Here we detect weak sequence similarity between the N- and C-terminal halves of the proteins using a sensitive method which uses a database containing the N- and C-terminal halves of all the sequences in ST3 and involves blast searches of each sequence in the database against the whole database. Unrelated families of secondary transporters of the same length and composition were used as controls. The sequence similarity involved major parts of the N- and C-terminal halves and not just a small stretch. The membrane topology of the homologous N- and C-terminal domains was deduced from the experimentally determined topology of the members of the 2HCT family. The domains consist of five transmembrane segments each and have opposite orientations in the membrane. The N terminus of the N-terminal domain is extracellular, while the N terminus of the C-terminal domain is cytoplasmic. The loops between the fourth and fifth transmembrane segment in each domain are well conserved throughout the class and contain a high fraction of residues with small side chains, Gly, Ala and Ser. Experimental work on the citrate transporter CitS in the 2HCT family indicates that the loops are re-entrant or pore loops. The re-entrant loops in the N- and C-terminal domains enter the membrane from opposite sides (trans-re-entrant loops). The combination of inverted membrane topology and trans-re-entrant loops represents a new fold for secondary transporters and resembles the structure of aquaporins and models proposed for Na+/Ca2+ exchangers.     

The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii: analysis of conserved sequences in NtrA proteins

Summary The nucleotide sequence of the Azotobacter vinelandii ntrA gene has been determined. It encodes a 56916 Dalton acidic polypeptide (AvNtrA) with substantial homology to NtrA from Klebsiella pneumoniae (KpNtrA) and Rhizobium meliloti (RmNtrA). NtrA has been shown to act as a novel RNA polymerase sigma factor but the predicted sequence of AvNtrA substantiates our previous analysis of KpNtrA in showing no substantial homology to other known sigma factors. Alignment of the predicted amino acid sequences of AvNtrA, KpNtrA and RmNtrA identified three regions; two showing>50% homology and an intervening sequence of <10% homology. The predicted protein contains a short sequence near the centre with homology to a conserved region in other sigma factors. The C-terminal region contains a region of homology to the subunit of RNA polymerase (RpoC) and two highly conserved regions one of which is significantly homologous to known DNA-binding motifs. In A. vinelandii, ntrA is followed by another open reading frame (ORF) which is highly homologous to a comparable ORF downstream of ntrA in K. pneumoniae and R. meliloti.     

Evidence that the Bacillus subtilis SpoIIGA protein is a novel type of signal-transducing aspartic protease

The bacterium Bacillus subtilis undergoes endospore formation in response to starvation. sigma factors play a key role in spatiotemporal regulation of gene expression during development. Activation of sigma factors is coordinated by signal transduction between the forespore and the mother cell. sigma(E) is produced as pro-sigma(E), which is activated in the mother cell by cleavage in response to a signal from the forespore. We report that expression of SpoIIR, a putative signaling protein normally made in the forespore, and SpoIIGA, a putative protease, is necessary and sufficient for accurate, rapid, and abundant processing of pro-sigma(E) to sigma(E) in Escherichia coli. Modeling and mutational analyses provide evidence that SpoIIGA is a novel type of aspartic protease whose C-terminal half forms a dimer similar to the human immunodeficiency virus type 1 protease. Previous studies suggest that the N-terminal half of SpoIIGA is membrane-embedded. We found that SpoIIGA expressed in E. coli is membrane-associated and that after detergent treatment SpoIIGA was self-associated. Also, SpoIIGA interacts with SpoIIR. The results support a model in which SpoIIGA forms inactive dimers or oligomers, and interaction of SpoIIR with the N-terminal domain of SpoIIGA on one side of a membrane causes a conformational change that allows formation of active aspartic protease dimer in the C-terminal domain on the other side of the membrane, where it cleaves pro-sigma(E).     

Kinetic and regulatory properties of HK I(+), a modified form of the type I isozyme of mammalian hexokinase in which interactions between the N- and C-terminal halves have been disrupted

A modified form (HK I(+)) of rat Type I hexokinase (HK I) has been expressed. HK I(+) contains a centrally located polyalanine insert which, along with the known helical propensity of adjacent sequence, was expected to lead to alpha-helix formation, with resulting distension of the molecule and disruption of interactions between the N- and C-terminal halves. The properties of HK I(+) are consistent with this expectation and with previous proposals that (1) inhibition of HK I by Glc-6-P or its analogs and antagonism of this inhibition by P(i) result from competition of these ligands for a binding site in the N-terminal half of HK I, with resulting conformational changes propagated through interactions with the catalytic C-terminal half, and (2) binding of Glc-6-P to a site in the C-terminal half of HK I is obstructed by interactions between the halves, present in HK I but not HK I(+).