PubMed Health⌕ Search

Biomedical subjects

S Del Rio

Publications and source records attributed to S Del Rio.

7 recordsLinked to original sources
PubMed1996

Functional interactions between the zinc fingers of Xenopus transcription factor IIIA during 5S rRNA binding.

We have used a collection of mutant forms of Xenopus transcription factor IIIA (TFIIIA) to study its interaction with 5S rRNA. This collection includes a set of nine mutant proteins, each of which contains a structural disruption in one of the nine zinc fingers of TFIIIA (broken-finger mutants), and a pair of complementary N- and C-terminal truncation mutants. Equilibrium and kinetic binding analyses in conjunction with RNAse protection and interference assays have been used to characterize the RNA-protein interaction in each case. We find that alternative binding modes are available for specific, high-affinity recognition of 5S rRNA by TFIIIA. These binding modes are distinct kinetically and structurally, and the mode of recognition adopted by wild-type TFIIIA when binding to intact 5S rRNA is dependent on the structural integrity of zinc fingers 5 and 6 in TFIIIA and continuity of the sugar-phosphate backbone in loop A of 5S rRNA. Disruption of any of these components allows adoption of one or more alternative modes of binding. In the wild-type TFIIIA-5S rRNA complex, some portions of TFIIIA, most notably the N-terminal three zinc fingers, are prevented from interacting with 5S rRNA in an energetically optimal way, and instead adopt a mode of binding that represents a compromise with the rest of the protein.

Animals↗
PubMed1993

The function of individual zinc fingers in sequence-specific DNA recognition by transcription factor IIIA.

Mutations in Zn(2+)-coordinating histidine residues have been used to generate forms of transcription factor IIIA containing structural disruptions in single zinc fingers. These mutant proteins have been analyzed with respect to the structural and functional independence of individual zinc fingers in TFIIIA. They have also been used to assess the energetic contributions to binding and the sites of interaction of each of the nine zinc fingers of TFIIIA with the 5 S rRNA gene. The results are surprising and suggest a complex mode of binding in which the interactions of structurally independent zinc fingers with the DNA substrate are non-uniform and functionally interdependent in a way that may help to explain some of the unusual properties of TFIIIA.

Animals↗
PubMed1993

The role of zinc fingers in transcriptional activation by transcription factor IIIA.

We have described elsewhere a number of the properties of a set of mutant forms of Xenopus transcription factor IIIA (TFIIIA) containing single amino acid substitutions that result in the structural disruption of individual zinc finger domains. These "broken finger" proteins have now been analyzed with respect to their ability to support transcription of 5S rRNA genes in vitro. Disruption of any one of the first six zinc fingers of TFIIIA has no discernible effect on the activity of the protein in supporting 5S rRNA synthesis in standard in vitro transcription assays, despite the fact that some of these mutant proteins exhibit large decreases in their binding affinity for 5S rRNA genes in binary complexes. These results indicate that the activity of TFIIIA as a transcription factor can be largely independent of its equilibrium binding constant for the 5S rRNA gene in the absence of other components of the RNA polymerase III transcriptional apparatus. In fact, this finding is consistent with the known pathway and kinetics of assembly of 5S rRNA transcription complexes. In contrast to the results obtained with finger 1-6 mutants, analogous mutations in zinc fingers 7-9 of TFIIIA result in moderate to complete loss of transcriptional activity. We interpret these results to mean that the three C-terminal zinc fingers of TFIIIA are not only involved in binding to the internal control region of 5S rRNA genes but are also required, either directly or indirectly, for higher-order interactions that are important in transcription complex assembly, stability, or activity.

Animals↗
PubMed1993

Role of TFIIIA zinc fingers in vivo: analysis of single-finger function in developing Xenopus embryos.

The Xenopus 5S RNA gene-specific transcription factor IIIA (TFIIIA) has nine consecutive Cys2His2 zinc finger motifs. Studies were conducted in vivo to determine the contribution of each of the nine zinc fingers to the activity of TFIIIA in living cells. Nine separate TFIIIA mutants were expressed in Xenopus embryos following microinjection of their respective in vitro-derived mRNAs. Each mutant contained a single histidine-to-asparagine substitution in the third zinc ligand position of an individual zinc finger. These mutations result in structural disruption of the mutated finger with little or no effect on the other fingers. The activity of mutant proteins in vivo was assessed by measuring transcriptional activation of the endogenous 5S RNA genes. Mutants containing a substitution in zinc finger 1, 2, or 3 activate 5S RNA genes at a level which is reduced relative to that in embryos injected with the message for wild-type TFIIIA. Proteins with a histidine-to-asparagine substitution in zinc finger 5 or 7 activate 5S RNA genes at a level that is roughly equivalent to that of the wild-type protein. Zinc fingers 8 and 9 appear to be critical for the normal function of TFIIIA, since mutations in these fingers result in little or no activation of the endogenous 5S RNA genes. Surprisingly, proteins with a mutation in zinc finger 4 or 6 stimulate 5S RNA transcription at a level that is significantly higher than that mediated by similar concentrations of wild-type TFIIIA. Differences in the amount of newly synthesized 5S RNA in embryos containing the various mutant forms of TFIIIA result from differences in the relative number and/or activity of transcription complexes assembled on the endogenous 5S RNA genes and, in the case of the finger 4 and finger 6 mutants, result from increased transcriptional activation of the normally inactive oocyte-type 5S RNA genes. The remarkably high activity of the finger 6 mutant can be reproduced in vitro when transcription is carried out in the presence of 5S RNA. Disruption of zinc finger 6 results in a form of TFIIIA that exhibits reduced susceptibility to feedback inhibition by 5S RNA and therefore increases the availability of the transcription factor for transcription complex formation.

Animals↗
PubMed1983

Sweat chloride concentration in adults with pulmonary diseases.

In order to determine whether a high proportion of adults with pulmonary diseases have sweat chloride concentrations in the range usually considered diagnostic for cystic fibrosis (greater than 60 mEg/L), we performed the standard diagnostic "sweat test" of Gibson and Cooke prospectively on 187 subjects 18 to 85 yr of age who did not have cystic fibrosis (166 of them had some pulmonary or allergic disorder, and 21 were healthy). In this group, 99% had sweat chloride concentration less than 70 mEq/L, and 96%, less than 60 mEq/L. Those taking steroids had sweat chloride concentration slightly but significantly lower than those who did not take steroids, probably because of the mineralocorticoid effect. Six patients had pancreatitis, and 2 of those had sweat chloride concentration greater than 60 mEq/L, a distribution of values significantly different (p less than 0.005) from the rest of the population. Our results suggest that a very small proportion of adults with pulmonary diseases have sweat chloride concentrations in the range usually considered diagnostic for cystic fibrosis, and that the sweat test is a good discriminant for cystic fibrosis even in the adult age group.

Adolescent↗