Biomedical subjects

L Olvera

Publications and source records attributed to L Olvera.

3 recordsLinked to original sources

PubMed2000

Reciprocal domain evolution within a transactivator in a restricted sequence space.

offhough the concept of domain merging and shuffling as a major force in protein evolution is well established, it has been difficult to demonstrate how domains coadapt. Here we show evidence of coevolution of the Sinorhizobium meliloti NifA (SmNifA) domains. We found that, because of the lack of a conserved glycine in its DNA-binding domain, this transactivator protein interacts weakly with the enhancers. This defect, however, was compensated by evolving a highly efficient activation domain that, contrasting to Bradyrhizobium japonicum NifA (BjNifA), can activate in trans. To explore paths that lead to this enhanced activity, we mutagenized BjNifA. After three cycles of mutagenesis and selection, a highly active derivative was obtained. Strikingly, all mutations changed to amino acids already present in SmNifA. Our artificial process thus recreated the natural evolution followed by this protein and suggests that NifA is trapped in a restricted sequence space with very limited solutions for higher activity by point mutation.

Amino Acid Sequence↗

PubMed1998

In vivo genomic footprinting analysis reveals that the complex Bradyrhizobium japonicum fixRnifA promoter region is differently occupied by two distinct RNA polymerase holoenzymes.

The Bradyrhizobium japonicum fixRnifA operon is transcribed from two promoters: fixRp1, a -24/-12 promoter recognized by the sigma54-holoenzyme form of the RNA polymerase, and fixRp2, a -35/-10 promoter that is transcribed by a second, unidentified, form of RNA polymerase holoenzyme. The fixRp1 promoter is autoregulated during microaerobiosis by NifA, whereas fixRp2 is also activated, but by a different regulatory protein. The main transcription start sites for these promoters are just two nucleotides apart, such that the conserved -12 and -10 regions of fixRp1 and fixRp2, respectively, must overlap each other, whereas the -24 and -35 regions lie one DNA helical turn apart. Using in vivo genomic dimethyl sulfate and KMnO4 footprinting, we showed that the promoter region is differentially protected, depending upon which holoenzyme is bound. Mutagenesis analyses indicated that positions from -12 to -14 are critical for the activity of both promoters, whereas mutations at -10 and -11 affected mainly fixRp2 expression. When the sequence of the putative -35 region of fixRp2 was modified to match the putative consensus, expression from this promoter was increased 3-fold and the reactivity toward KMnO4, but not the transcriptional start site, moved two nucleotides further upstream, indicating that the altered promoter forms a different open complex. Additionally, we detected NifA-dependent methylation protection of two atypical NifA binding sites and protection of guanine -75. The latter residue is located in a region critical for fixRp2 promoter activation. The results present direct physical evidence of the complexity of the organization, regulation, and function of the fixRnifA promoter region.

Bacterial Proteins↗

PubMed1998

In vivo studies on the positive control function of NifA: a conserved hydrophobic amino acid patch at the central domain involved in transcriptional activation.

The eubacterial enhancer-binding proteins activate transcription by binding to distant sites and, simultaneously, contacting the RNA polymerase r54 promoter complex (Esigma54). The positive control function is located at the central domain of these proteins, but it is not know which specific region has the determinants for the interaction with Esigma54. Here, we present genetic evidence that a small region of hydrophobic amino acids, previously denominated C3, at the central domain of Bradyrhizobium japonicum NifA is involved in positive control. We obtained 26 missense mutants along this conserved region. Among these, only strains expressing the NifA(F307-->Y) and NifA(A310-->S) mutant proteins retained some of the transcriptional activity (<20%), whereas those carrying NifA(E298-->D) and NifA(T308-->S) had very low but detectable activity (< 1.0%). The rest of the NifA mutants did not induce any measurable transcriptional activity. When expressed in the presence of wild-type NifA, the great majority of the mutants displayed a dominant phenotype, suggesting that their oligomerization determinants were not altered. In vivo dimethyl-sulphate footprinting experiments for a subset of the NifA mutants showed that they were still able to bind specifically to DNA. Analysis of intragenic supressors highlight the functional role of a hydroxyl group at position 308 to activate transcription.

Amino Acid Sequence↗