Sara Austin

Role of metal ions in negative regulation of nitrogen fixation by the nifL gene product from Klebsiella pneumoniae

SummaryThe ability of the Klebsiella pneumoniae nifL gene product to antagonise NIFA mediated transcriptional activation from the nifH promoter in vivo was inhibited either by metal deprivation, or by the presence of the iron chelators EDDA or Desferal in the growth medium. This inhibition of the repressive activity of NIFL was reversed by the addition of ferrous or manganous ions to the medium but was unaffected by other transition metals. The dependence on metal ions for NIFL activity was observed when NIFL was overexpressed and when cultures were exposed to oxygen or high levels of fixed nitrogen. Immunochemical evidence suggests that NIFL and NIFA associate to form a functional protein complex. Metal ions are apparently not required for the formation of this complex.

The role of activator binding sites in transcriptional control of the divergently transcribed nifF and nif LA promoters from Klebsiella pneumoniae

The regulatory region spanning the divergently transcribed nif F and nifLA promoters contains a NIFA‐specific upstream activator sequence (UAS) located around +59, and two NTRC binding sites centred at −142 and −163 with respect to the nifLA transcription start site. We have constructed mutations in each of these binding sites and examined their role in transcriptional activation of the divergently transcribed promoters. Analysis of a mutation at +60 confirms that the UAS is required for efficient NIFA‐mediated activation of nifF transcription. This sequence is also required for maximal activation of the nifLA promoter. Mutations at −169 and −148, within the two NTRC binding sites, reduce activation of the nifLA promoter by NTRC in vivo and lower the affinity of the activator for these sites in vitro. Phosphorylation of NTRC by NTRB is required for efficient binding of NTRC to these sites.

Core RNA polymerase assists binding of the transcription factor σ;54 to promoter DNA

The Sigma subunit of bacterial RNA polymerase Is necessary for the specific binding of RNA polymerase holoenzyme to promoter DNA. Promoter complexes which form with holoenzyme containing σ;54 remain as closed complexes unless they are activated by one class of enhancer binding protein. The σ;54 transcription factor can bind specifically to certain promoter sites in the absence of the core RNA polymerase subunits. This property has allowed demonstration of a new role for core polymerase in transcription, namely that it assists the binding of σ;54 to promoter DNA, An altered form of σ;54 with a deletion within the amino‐terminal region showed increased affinity for specific DNA‐binding sites. Although able to complex with core RNA polymerase the mutant σ;54 failed to respond to core polymerase in the manner characteristic of the wild‐type σ;54 by altering its footprint. This result indicates that σ;54 has a latent DNA‐binding activity which is revealed by core RNA polymerase, and possibly involves a change in σ;54 conformation. Promoter complexes which formed with σ;54‐holoenzyme appeared to be qualitatively different, depending upon the target promoter sequence, suggesting that different activatable complexes form at different promoter sequences.

Requirements for transcriptional activation in vitro of the nitrogen‐regulated gInA and nifLA promoters from Klebsiella pneumoniae: dependence on activator concentration

Three proteins involved in nitrogen regulation In Klebsiella pneumoniae, NTRA, NTRB and NTRC, have been purified. In a defined in vitro system all three NTR proteins are required for initiation of transcription at the ntr activatable glnA and nifLA promoters. However, in crude S‐30 extracts, transcription from the glnA promoter, but not the nifLA promoter, can be activated in the absence of NTRB. A higher concentration of NTRC is required for activation of nifLA transcription than for glnA transcription. Sequences located between ‐227 and ‐158 with respect to the nifL transcription start site are required for efficient activation of the nifLA promoter in vitro.

DNA supercoiling response of the σ54-dependent Klebsiella pneumoniae nifL promoter in vitro

Transcription from the σ54-dependent Klebsiella pneumoniae nifL and glnAp2 promoters is activated by the general nitrogen regulatory protein NTRC. Unlike the glnAp2 promoter, which is relatively insensitive to changes in DNA supercoiling, transcription from nifL in vitro in a chloride-based buffer is supercoiling-dependent at physiological salt concentrations. The replacement of chloride with an acetate-based buffer decreases the stringency of the nifL supercoiling response, but open complexes formed on linear nifL promoter DNA under these conditions are unstable and less extensive than those found on supercoiled (form I) DNA. We have introduced mutations in particular elements of the nifL promoter that increase its homology to glnAp2. At the wild-type nifL promoter, σ54-RNA polymerase makes only limited contacts with the promoter in the absence of NTRC. However, a G to T change at −26 (nifL74) allows the formation of a stable closed complex with σ54-holoenzyme on both linear and form I templates in the absence of the activator. The combination of C to T mutations at −3 and −1 (nifL18) increases the A + T rich nature of the melted region and stabilizes open complexes formed on linear DNA. Open complex formation as a function of superhelical density was assessed at each promoter. Formation of open complexes at glnAp2 peaks at −0.024 and declines at higher superhelical densities, whereas at the wild-type nifL promoter, open complex formation peaks at −0.067 and is not detectable at superhelical densities less than −0.032. Both the nifL74 and nifL18 mutations altered the supercoiling response, increasing the ability to form open complexes at low superhelical densities. The presence of the nifL74 and nifL18 mutations in combination further altered the response of the promoter to DNA supercoiling. These observations suggest that the promoter as a whole, and not any one promoter element, mediates the transcriptional response to DNA supercoiling.

Substitutions at a single amino acid residue in the nitrogen‐regulated activator protein NTRC differentially influence its activity in response to phosphorylation

Four substitutions at serine residue 160 which increase the activity of the σ54‐dependent activator protein NTRC in the absence of NTRB have been analysed in detail. Mutagenesis of the putative phosphoacceptor site of NTRC and analysis of double mutants indicate that the positive control function of the S160W and S160C mutants is phosphorylation‐dependent, whereas the activity of the S160Y and S160F mutants is phosphorylation‐independent. This was confirmed with two purified mutant proteins in vitro. Occupancy of tandem NTRC‐binding sites upstream of the Klebsiella pneumoniae nifL promoter by S160W protein is also phosphorylation‐dependent in contrast to occupancy by S160F protein, confirming that both the DNA‐binding and activator functions of NTRC are influenced by phosphorylation. The S160W and S160C mutants are apparently more responsive than wild‐type protein to ‘cross‐talk’ by other members of the histidine protein kinase family but are less responsive to phosphorylation and dephosphorylation mediated by NTRB.

Defective initiation on natural messenger RNA by cell free systems from Krebs ascites cells incubated at elevated temperatures

1. Cell-free systems prepared from Krebs II ascites cells incubated at 45 degrees C have a much lower endogenous activity than those from cells incubated at 37 degrees C. The endogenous activity is mainly due to completion of polypeptide chains initiated in the intact cell. The low activity of the 45 degrees C system is due to a lesion in initiation in cells incubated at 45 degrees C. 2. Cell-free systems from cells incubated at 45 degrees C can translate efficiently poly (U) at 8 mM Mg2+. However, they initiate poorly on globin mRNA, indicating that these systems reflect the situation in the intact cell. 3. The lesion in globin mRNA translation in 45 degrees C systems can be overcome by addition of reticulocyte initiation factors. At saturation concentrations of factors, the response of a 45 degrees C system is restored to almost normal. 4. 45 degrees C systems from 40-S initiation complexes with methionyl tRNAfmet almost as efficiently as normal, but their ability for form 80-S complexes with globin mRNA is impaired, unless they are supplied with exogenous initiation factors.

PURIFICATION AND ACTIVITIES OF THE RHODOBACTER CAPSULATUS RPON (SIGMA N) PROTEIN

The rpoN‐encoded sigma factors (σN) are a distinct class of bacterial sigma factors, with no obvious homology to the major σ70 class. The σN‐containing RNA polymerase holoenzyme functions in enhancer‐dependent transcription to allow expression of positively controlled genes. We have purified the Rhodobacter capsulatusσN protein, which is distinctive in lacking an acidic region implicated in the melting of promoter DNA by the Escherichia coliσN holoenzyme, and may represent a minor subclass of σN proteins. Assays of promoter recognition and holoenzyme formation and function showed that the purified R. capsulatusσN protein is distinct in activity compared to the enteric proteins, but retains the broad functions described for these proteins. As first described for the Kleb‐siella pneumoniae protein, promoter recognition in the absence of core RNA polymerase was detected, but contact of certain promoter bases by the R. capsulatusσN protein and its response to core RNA polymerase was clearly different from that determined for the K. pneumoniae and E. coli proteins. Results are discussed in the context of a requirement to modulate the activity of the DNA‐binding surfaces of σN to regulate σN function. Circular dichroism was used to evaluate the structure of the R. capsulatus protein and revealed differences in the tertiary signals as compared to the K. pneumoniae protein, some of which are attributable to the DNA‐binding domain of σN

Identification and characterization of a biosynthetic precursor (preprolactin) of ovine pituitary prolactin.

Poly(A)-containing mRNA prepared from ovine anterior pituitary glands stimulated the incorporation of radioactivity into protein in a wheat germ cell-free system from 7- to 25-fold over background. Approximately 30% of the total protein synthesized could be precipitated by antiserum to prolactin, indicating that a molecule which shares antigenic determinants with prolactin was being synthesized. Analysis of the immunoprecipitable cell-free translation product, by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulphate demonstrated the presence of a prolactin-like molecule with a molecular weight greater than that of prolactin itself. Partial N-terminal sequence analysis of the immunoprecipitable translation product showed that it contained one methionine residue at position 1 and several leucine residues within the first 30 amino acids. Experiments with labelled initiator and internal met tRNAMet indicated that the product being synthesized contained the initiating methionine and was probably the initial translation product of prolactin mRNA.

Regulation of Nitrogen Fixation by the NIFL and NIFA Proteins from Azotobacter Vinelandii

The extreme oxygen sensitivity of the nitrogenase component proteins and the high energetic requirements for nitrogen fixation impose physiological constraints on diazotrophy necessitating stringent regulation of nif gene transcription in response to fixed nitrogen and environmental oxygen (Hill, 1992). In most diazotrophs expression of genes required for the synthesis of molybdenum nitrogenase is positively controlled by the transcriptional activator NIFA, an enhancer binding protein which catalyses the formation of open promoter complexes by the holoenzyme form of RNA polymerase containing the alternative sigma factor σN (EσN) (Morett and Segovia 1993). Binding sites for NIFA, termed Upstream Activator Sequences (UAS) are usually located more than 100 base pairs upstream of the transcription start site and the activator appears to make contact with downstream bound EσNvia the formation of a DNA loop (Buck et al 1987). The formation of such loops is facilitated by the binding of an auxilliary protein, Integration Host Factor (IHF) which may stabilise these structures by correctly orientating the activator and polymerase binding sites and offsetting the bending energy required for DNA loop formation (Hoover et al 1990). Productive interactions between NIFA and EσN promote the transition from the closed promoter complex to the open promoter complex. An unusual feature of this reaction with respect to prokaryotic transcription systems is that it requires the hydrolysis of a nucleoside triphosphate, principally ATP or GTP (Popham et al 1989; Berger et al 1994; Austin et al 1994) (Fig 1)