Tight binding of the 5' exon to domain I of a group II self-splicing intron requires completion of the intron active site.
Group II self-splicing requires the 5' exon to form base pairs with two stretches of intronic sequence (EBS1 and EBS2) which also bind the DNA target during retrotransposition of the intron. We have used dimethyl sulfate modification of bases to obtain footprints of the 5' exon on intron Pl.LSU/2 from the mitochondrion of the alga Pylaiella littoralis, as well as on truncated intron derivatives. Aside from the EBS sites, which are part of the same subdomain (ID) of ribozyme secondary structure, three distant adenines become either less or more sensitive to modification in the presence of the exon. Unexpectedly, one of these adenines in subdomain IC1 is footprinted only in the presence of the distal helix of domain V, which is involved in catalysis. While the loss of that footprint is accompanied by a 100-fold decrease in the affinity for the exon, both protection from modification and efficient binding can be restored by a separate domain V transcript, whose binding results in its own, concise footprint on domains I and III. Possible biological implications of the need for the group II active site to be complete in order to observe high-affinity binding of the 5' exon to domain I are discussed. (+info)
Effects of inhibitors and substitutes for chloride in lumen on p-aminohippurate transport by isolated perfused rabbit renal proximal tubules.
The transport step for p-aminohippurate (PAH) from cell to lumen across the luminal membrane of rabbit proximal tubules has not been adequately defined. To examine this process more closely, we determined the effects of possible transport inhibitors and substitutes for chloride on PAH secretion in isolated perfused S2 segments of rabbit proximal tubules. The addition of 4-acetamido-4'-isothiocyano-2,2' disulfonic stilbene (10(-4) M) to the perfusate irreversibly inhibited PAH secretion, whereas the addition of probenecid (10(-4) M) to the perfusate reversibly inhibited PAH secretion. PAH secretion was unaffected by thiocyanate replacement of chloride in the luminal perfusate, reversibly inhibited by 15 to 20% by methyl sulfate replacement, and irreversibly inhibited by isethionate replacement. Because the luminal membrane is at least as permeable to thiocyanate as to chloride, less permeable to methyl sulfate, and much less permeable to isethionate, these data suggest that the PAH transport step from cells to lumen does not require chloride in the lumen but does require a highly permeant anion. During inhibition of PAH transport from cells to lumen, PAH uptake across the basolateral membrane was also reduced, suggesting some type of feedback inhibition. The data are compatible with PAH transport across the luminal membrane by an anion exchanger, a potential-driven uniporter, both carriers, or a carrier that can function in both modes. (+info)
Structural characterization of DNA and RNA sequences recognized by the gene 5 protein of bacteriophage fd.
The single-stranded DNA sequence d(GT5G4CT4C) occurs close to the origin of replication within the intergenic region of the viral strand of bacteriophage fd. The RNA analogue of this sequence r(GU5G4CU4C) forms part of the untranslated leader sequence of the gene 2 mRNA and is specifically bound by the fd gene 5 protein in its role as a translational repressor. The structure of these sequences is likely to have an important role in the control of both DNA replication and RNA translation in the phage. We show that this 16 nt sequence, in both a DNA and an RNA context, can exist in a structured and unstructured form as determined by high-resolution gel filtration and non-denaturing gel electrophoresis. The CD spectrum of the structured form is characteristic of parallel guanine tetraplexes. The structured form of the DNA sequence melts at approx. 47 degrees C in the presence of Na+ ions but the structure is stabilized up to 75 degrees C in the presence of K+ ions. The RNA structure is more stable than the equivalent DNA structure (melting temperature approx. 62 degrees C), and its stability is further enhanced in the presence of K+ ions. Two of the central guanine residues are fully protected from cleavage as determined by dimethyl sulphate protection experiments, whereas methylation interference studies show that methylation of any of the four central guanine residues inhibits structure formation. Our results demonstrate that the structured form of the nucleic acid is mediated through the formation of a guanine-tetraplex core region, in RNA this might be further stabilized by the presence of weaker uracil quartets. (+info)
Common components of patch-clamp internal recording solutions can significantly affect protein kinase A activity.
Common components of whole-cell internal recording solutions were tested both in vitro and in patch-clamp experiments for their effects on the activity of cAMP-dependent protein kinase. Potassium fluoride (KF), 440 mM trimethylamine chloride and exclusion of bovine serum albumin (BSA) decreased the activity of the enzyme, while ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) and the potassium salts of aspartate, gluconate, methylsulfate and monobasic phosphate increased its activity. Addition of KF to the internal solution produced a hyperpolarizing shift in the V1/2 of Ih channel activation, consistent with the KF-induced reduction of protein kinase A activity. Therefore, consideration of the composition of internal solutions is warranted when studying channel physiology by patch-clamp techniques. (+info)
Transcription of the human U2 snRNA genes continues beyond the 3' box in vivo.
The 3' box of the human class II snRNA genes is required for proper 3' processing of transcripts, but how it functions is unclear. Several lines of evidence suggest that termination of transcription occurs at the 3' box and the terminated transcript is then a substrate for processing. However, using nuclear run-on analysis of endogenous genes, we demonstrate that transcription continues for at least 250 nucleotides beyond the 3' box of the U2 genes. Although in vivo footprinting analysis of both the U1 and U2 genes detects no protein-DNA contacts directly over the 3' box, a series of G residues immediately downstream from the 3' box of the U1 gene are clearly protected from methylation by dimethylsulfate. In conjunction with the 3' box of the U1 gene, this in vivo footprinted region causes termination of transcription of transiently transfected U2 constructs, whereas a 3' box alone does not. Taken together, these results indicate that the 3' box is not an efficient transcriptional terminator but may act as a processing element that is functional in the nascent RNA. (+info)
Sulfation of chondroitin sulfate in human articular cartilage. The effect of age, topographical position, and zone of cartilage on tissue composition.
The chondroitin ABC lyase digestion products of normal human femoral condyle articular cartilage and of purified aggrecan were analyzed for their mono- and nonsulfated disaccharide composition. Changes in the total tissue chemistry were most pronounced during the period from birth to 20 years of age, when the -[GlcAbeta,3GalNAc6]- disaccharide content increased from approximately 50% to 85% of the total disaccharide content and there was a concomitant decrease in the content of the 4-sulfated disaccharide. In general, the disaccharide content of the deeper layers of immature cartilage were richer in the 4-sulfated residue than the upper regions of the tissue. As the tissue aged and decreased in thickness, the disaccharide composition became more evenly 6-sulfated. The newly synthesized chondroitin sulfate chains had a similar composition to the endogenous chains and also underwent the same age and zonal changes. The monoclonal antisera 3B3(+) and 2B6(+) were used to immunolocalize the unsaturated 6- and 4-sulfated residues generated at the reducing termini of the chondroitin sulfate chains by digestion with chondroitin ABC lyase, and these analyses indicated that the sulfation pattern at this position did not necessarily reflect the internal disaccharide composition of the chains. In summary, the sulfation pattern of chondroitin sulfate disaccharides from human normal articular cartilage varies with the age of the specimen, the position (topography) on the joint surface, and the zone of cartilage analyzed. Furthermore, these changes in composition are a consequence of both extracellular, post-translational processing of the core protein of aggrecan and changes in the sulfotransferase activity of the chondrocyte. (+info)
Importance of a 5' stem-loop for longevity of papA mRNA in Escherichia coli.
High-level expression of the major pilus subunit (PapA) of uropathogenic strains of Escherichia coli results in part from the unusually long lifetime of the mRNA that encodes this protein. Here we report that the longevity of papA mRNA derives in large measure from the protection afforded by its 5' untranslated region. This papA RNA segment can prolong the lifetime of an otherwise short-lived mRNA to which it is fused. In vivo alkylation studies indicate that, in its natural milieu, the papA message begins with a stem-loop structure. This stem-loop is important for the stabilizing effect of the papA 5' untranslated region, as evidenced by the significant acceleration in papA mRNA decay that results from its removal. (+info)
Basis for prokaryotic specificity of action of aminoglycoside antibiotics.
The aminoglycosides, a group of structurally related antibiotics, bind to rRNA in the small subunit of the prokaryotic ribosome. Most aminoglycosides are inactive or weakly active against eukaryotic ribosomes. A major difference in the binding site for these antibiotics between prokaryotic and eukaryotic ribosomes is the identity of the nucleotide at position 1408 (Escherichia coli numbering), which is an adenosine in prokaryotic ribosomes and a guanosine in eukaryotic ribosomes. Expression in E.coli of plasmid-encoded 16S rRNA containing an A1408 to G substitution confers resistance to a subclass of the aminoglycoside antibiotics that contain a 6' amino group on ring I. Chemical footprinting experiments indicate that resistance arises from the lower affinity of the drug for the eukaryotic rRNA sequence. The 1408G ribosomes are resistant to the same subclass of aminoglycosides as previously observed both for eukaryotic ribosomes and bacterial ribosomes containing a methylation at the N1 position of A1408. The results indicate that the identity of the nucleotide at position 1408 is a major determinant of specificity of aminoglycoside action, and agree with prior structural studies of aminoglycoside-rRNA complexes. (+info)