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P A Sharp

Publications and source records attributed to P A Sharp.

At least 73 records · Page 4Linked to original sources
PubMed1994

Dynamic association of proteins with the pre-mRNA branch region.

The association of proteins with the branch site region during pre-mRNA splicing was probed using a novel methodology to site-specifically modify the pre-mRNA with the photo-reagent benzophenone. Three sets of proteins were distinguished by the kinetics of their associations with pre-mRNAs, by their association with discrete splicing complexes, and by their differing factor requirements. An early U1 snRNP-dependent cross-link of the branch region to a p80 species was followed by cross-links to p14, p35, and p150 polypeptides associated with the U2 snRNP-pre-mRNA complex. Concomitant with formation of the spliceosome, a rearrangement of protein factors about the branch region occurred, in which the p35 and p150 cross-links were replaced by p220 and p70 species. These results establish that the branch region is recognized in a dynamic fashion by multiple distinct proteins during the course of spliceosomal assembly.

Base Sequence↗
PubMed1994

Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP.

Individual small nuclear ribonucleoproteins (snRNPs) U1, U2, and U4/U6 were removed from nuclear extracts of HeLa cells by antisense affinity depletion. Addition of a highly purified preparation of SR proteins fully restored splicing activity in reactions depleted of U1 snRNP but did not reconstitute splicing in reactions depleted of the other snRNPs. Affinity selection experiments revealed that spliceosomes lacking U1 snRNA formed in the U1 snRNP-depleted reactions reconstituted with SR proteins. Thus, high concentrations of SR proteins facilitate the assembly of precursor messenger RNA (pre-mRNA) into a spliceosome in the absence of interactions with U1 snRNP.

Base Sequence↗
PubMed1994

Homeodomain determinants of major groove recognition.

The homeodomain is a highly conserved structural module that binds DNA and participates in protein-protein interactions. Most homeodomains contain residues at positions 47 and 51 which mediate recognition of a TAAT core binding sequence in the major groove. The constraints imposed on the identity of these residues by homeodomain structure and DNA docking have been examined in the context of the POU domain of the Oct-1 transcription factor. A bacterial library, in which POU homeodomain residues 47 and 51 have been randomized, was probed on nitrocellulose filters for the binding of DNA fragments containing the consensus octamer sequence. The residues which provide for the highest affinity interaction with the octamer consensus sequence, and the greatest specificity, are the highly conserved wild-type residues valine 47 and asparagine 51. Interestingly, a class of variants containing arginine at position 51 was also detected in the screen and found to have moderate affinity for the consensus sequence but reduced specificity compared to the wild-type protein. A single variant containing arginine at both positions 47 and 51 was detected when the library was probed with fragments containing nucleotide substitutions at positions expected to be contacted by residues 47 and 51. This variant was used to alter the DNA-binding specificity of a transcriptional regulatory complex which depends upon Oct-1 for DNA recognition. These findings suggest that homeodomain structure and DNA docking constrain in the versatility of the domain in that only a limited set of amino acid determinants can endow the domain with specific, high-affinity DNA binding.

Base Sequence↗
PubMed1994

Multiple sets of basal factors initiate transcription by RNA polymerase II.

The minimal requirements for transcription initiation from supercoiled templates were determined for the two major forms of TATA-binding factors found in cell extracts, the 300-kDa B-TFIID and the 1000-kDa D-TFIID complexes. As had been observed for the TATA-binding protein (TBP) subunit (Parvin and Sharp, 1993), transcription from the IgH promoter minimally requires TFIID activity plus TFIIB and RNA polymerase II. This minimal reaction is only active on negatively supercoiled template DNA. In contrast, the supercoiled templates encoding the adenovirus major late promoter (MLP), or several other promoters, require the addition of TFIIF to the minimal reaction. Further addition of TFIIE and TFIIH boosts the level of transcription from these latter promoters but is not required. In contrast to the complete reaction on linear template, transcription from supercoiled IgH or MLP templates does not require the hydrolysis of the beta-gamma bond of ATP. Fourteen different core promoters were compared in complete and minimal basal transcription reactions reconstituted with one of the three TATA activities: TBP, B-TFIID, and D-TFIID. Of these 14 promoters, only the IgH was active in the absence of TFIIF, and the other promoters demonstrated different levels of transcription depending on which basal factors were present in reaction. It is proposed that a significant level of basal transcription only requires a minimal set of factors, and stimulation by upstream activators may in part be mediated by the inclusion of additional basal factors into the initiation reaction.

Animals↗
PubMed1994

Branch nucleophile selection in pre-mRNA splicing: evidence for the bulged duplex model.

Selection of the nucleophile for the first step of nuclear pre-mRNA splicing was probed by site-specific incorporation into splicing substrates of nucleotides modified at the 2' position. The differing abilities of ribose, 2'-deoxyribose, and arabinose nucleotides to base-pair within an RNA.RNA duplex and to contribute a nucleophilic 2'-OH group were exploited to analyze the paired/unpaired disposition of the branch site nucleotide. The results provide direct evidence for a bulged duplex model in which either of two adjacent purines within the consensus branch site sequence may shift into a bulged position and contribute the 2'-OH group for the first step of splicing. Furthermore, the presence of a consensus branch site that cannot present a reactive nucleophile suppresses splicing, including the use of cryptic branch sites elsewhere. We conclude that the branch site region base-pairing with U2 snRNA determines the first step nucleophile and persists at the time of the first transesterification reaction.

Arabinose↗
PubMed1994

The interaction of GATA-binding proteins and basal transcription factors with GATA box-containing core promoters. A model of tissue-specific gene expression.

The core promoters of the rat platelet factor 4 (PF4), mouse erythropoietin and chicken beta globin genes contain a GATA motif in place of the consensus TATAAA site. In the case of the PF4 gene, this site has been shown to play a critical role in restricting transcription to the megakaryocyte lineage. In order to understand the mechanism of tissue specificity, we investigated the function of the GATA box-containing promoters in vitro. Our studies show that the TATA-binding protein of TFIID is required for initiation of transcription from the GATA box-containing promoters. GATA-1 interacts with the core promoter GATA motif and inhibits generation of preinitiation complexes. The functional significance of the inhibition of preinitiation complexes is supported by in vitro transcription assays in which transcription from the PF4 and erythropoietin core promoters is suppressed by GATA-1. We also demonstrate that GATA-2 inhibits initiation of transcription from the PF4 core promoter. Based on these results, we propose a model in which repression of PF4 expression in nonmegakaryocytes is mediated, in part, by competition between GATA-binding proteins and basal factors for the core promoter.

Animals↗
PubMed1994

Association of nuclear matrix antigens with exon-containing splicing complexes.

mAbs raised against the human nuclear matrix (anti-NM)1 mAbs have been used to investigate the role of nuclear matrix antigens in pre-mRNA processing. The three anti-NM mAbs used in this study recognize antigens that are highly localized to nuclear matrix speckles. Surprisingly, all three of these mAbs preferentially immunoprecipitate splicing complexes containing exon sequences. The anti-NM mAbs efficiently immunoprecipitate the exon product complex but not complexes containing the lariat product after the second step of splicing. Two of the anti-NM mAbs completely inhibit pre-mRNA splicing in vitro. However, none of the anti-NM mAbs appear to recognize factors stably associated with splicing snRNPs. The three anti-NM mAbs predominantly react with distinct high molecular weight antigens, which belong to a class of nuclear proteins that selectively precipitate with Ser-Arg protein-splicing factors in the presence of high Mg2+ concentrations. Immunological, biochemical, and cell biological data indicate that two of the NM antigens are related to the defined set of Ser-Arg proteins. The results suggest the existence of an extended Ser-Arg family as a component of the nuclear matrix.

Amino Acid Sequence↗
PubMed1994

The role of cyclic AMP in the control of sugar transport across the brush-border and basolateral membranes of rat jejunal enterocytes.

Although the involvement of the adenylate cyclase system with glucose transport in the small intestine is poorly understood, there is increasing evidence that cyclic AMP stimulates sugar uptake. In order to study further the effects of cyclic AMP on this process, we have measured glucose accumulation by brush-border and basolateral membrane vesicles prepared from villus enterocytes following exposure of these cells to cyclic AMP and theophylline. Brush-border vesicles derived from enterocytes incubated with cyclic AMP and theophylline accumulated significantly more glucose over a wide range of sugar concentrations, suggesting a change in maximum velocity of the transport system. Glucose uptake by basolateral vesicles was increased at low, but not at high sugar concentrations. Incubation of isolated enterocytes with pancreatic glucagon at concentrations known to stimulate sugar transport by these cells significantly increased enterocyte levels of cyclic AMP. Treatment with glucagon or cyclic AMP resulted in significant hyperpolarization of the potential difference across the brush-border membrane, an important driving force for Na(+)-sugar cotransport. The response to glucagon and cyclic AMP appears to be caused by a decrease in Na+ permeability of the mucosal membrane. Taken together, these results suggest that cyclic AMP is a mediator of the actions of glucagon on enterocytes and provide further evidence for a role of cyclic AMP in the modulation of sugar transport across the intestinal enterocyte.

Animals↗
PubMed1994

The effect of rapid changes in plasma sugar concentration on the brush-border potential difference in rat jejunum.

The effect of infusion (2 h) of various sugars (20%, w/v) or early diabetes mellitus (2 h following injection of streptozotocin) on the potential difference (PD) across the brush border of rat jejunum has been studied in vitro. Infusion of glucose or galactose resulted in hyperpolarization of the brush-border PD (-55.2 and -54.9 mV respectively) compared with mannitol-infused animals (-47.7 mV). Infusion of fructose and alpha-methyl glucoside was without effect on PD. Ion substitution experiments showed that the glucose infusion-induced hyperpolarization was due either to a decrease in Cl- conductance and/or increased K+ conductance of the brush-border membrane. Na+ conductance appeared to be unaltered in these experiments. Since galactose infusion hyperpolarized PD without elevating plasma glucose or insulin levels, it seems that neither glucose nor insulin alone is responsible for the significantly greater PD. Treatment with streptozotocin produced glycosuria and hyperglycaemia within approximately 90 min and animals were used 30-60 min later. PD in streptozotocin-treated animals (-51.8 mV) was significantly greater than that measured in diluent-injected animals (-45.3 mV, P < 0.001). However, unlike the situation with sugar infusion, ion substitution experiments showed that the hyperpolarization seen in diabetic animals was due to a decreased Na+ conductance of the brush-border membrane. Since the magnitude of the brush-border PD dictates the electrochemical driving force for Na(+)-sugar cotransport, our data represent the first evidence for an effect of acute hyperglycaemia on the capacity for brush border sugar uptake. However, the enterocyte and systemic factors responsible for the membrane hyperpolarization are unclear.

Animals↗
PubMed1993

Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing.

Excision of introns from nuclear precursors to messenger RNAs (pre-mRNAs) by the spliceosome requires two distinct phosphodiester transfer (transesterification) reactions: exchange of a 3'-5' for a 2'-5' bond in the first step (lariat formation) and exchange of one 3'-5' phosphodiester for another in the second step (exon ligation). We report here determination of the stereochemical course of each step using splicing substrates that contained a chiral phosphorothioate. This has provided strong evidence that both steps occur as single 'in-line' SN2 nucleophilic displacement reactions, analogous to the mechanism of group I self-splicing introns. Additionally, because both steps are strongly inhibited by the RP phosphorothioate diastereomer, but not by SP, the spliceosome probably shifts between two active sites in catalysis of the two steps. Chemical and stereochemical similarities suggest that the catalytic site for the second step of spliceosomal processing is related to that of group I self-splicing introns.

Animals↗
PubMed1993

Purification of the major histocompatibility complex class I transcription factor H2TF1. The full-length product of the nfkb2 gene.

H2TF1 is a ubiquitous major histocompatibility complex (MHC) class I-specific transcription factor, which binds to the palindromic kappa B enhancer site upstream of MHC class I genes. Here we report that H2TF1 consists of a polypeptide with relative molecular mass 110,000, that corresponds to the predicted 100-kDa product (NF-kappa B2 p100) encoded by the candidate proto-oncogene nfkb2 (lyt-10). H2TF1 was purified by a novel affinity chromatography method and identified as the NF-kappa B2 p100 polypeptide by peptide sequencing as well as by reactivity with a specific antiserum. Purified H2TF1 binds the MHC kappa B site with high affinity (KD = 3 x 10(-11) M), in contrast with previous reports that NF-kappa B2 p100 did not bind DNA.

Amino Acid Sequence↗
PubMed1993

Crystal structure of yeast TATA-binding protein and model for interaction with DNA.

The C-terminal 179-aa region of yeast (Saccharomyces cerevisiae) TATA-binding protein (TBP), phylogenetically conserved and sufficient for many functions, formed crystals diffracting to 1.7-A resolution. The structure of the protein, determined by molecular replacement with coordinates from Arabidopsis TBP and refined to 2.6 A, differed from that in Arabidopsis slightly by an angle of about 12 degrees between two structurally nearly identical subdomains, indicative of a degree of conformational flexibility. A model for TBP-DNA interaction is proposed with the following important features: the long dimension of the protein follows the trajectory of the minor groove; two rows of basic residues conserved between the subdomains lie along the edges of the protein in proximity to the DNA phosphates; a band of hydrophobic residues runs down the middle of the groove; and amino acid residues whose mutation alters specificity for the second base of the TATA sequence are juxtaposed to that base.

Amino Acid Sequence↗
PubMed1993

The CRE-binding factor ATF-2 facilitates the occupation of the CCAAT box in the fibronectin gene promoter.

The cAMP response element (CRE) and the CCAAT box of the fibronectin gene promoter are separated by only twenty base pairs. A specific factor that binds the CRE interacts cooperatively with the protein which binds to the adjacent CCAAT box, stimulating transcription [1992, J. Biol. Chem. 267, 12767-12774]. Here we show that the CRE factor is an heterodimer between a 43 kDa and the '73 kDa' CRE-binding proteins and we identify the latter as ATF-2 (also named CRE-BPI), a protein implicated in recruiting transcriptional activators to promoters, able to form heterodimers with Jun and for which a sequence-deduced MW of 55 kDa had been previously reported.

Activating Transcription Factors↗
PubMed1993

DNA topology and a minimal set of basal factors for transcription by RNA polymerase II.

Immunoglobulin heavy chain (IgH) gene transcription in vitro can be reconstituted with a minimal reaction containing only TATA-binding protein (TBP), TFIIB, and RNA polymerase II (pol II) when the template is negatively supercoiled. Transcription from linear DNA templates containing either the IgH or the adenovirus major late promoters (MLPs) requires in addition TFIIF, TFIIE, TFIIH, and a fraction containing TFIIA and TFIIJ. Promoters vary in their activities in the minimal reaction. Initiation at the adenovirus MLP site was not observed in this reaction, even with templates containing negative superhelical density. When only TBP, TFIIB, and pol II were present in the reaction, the more negatively supercoiled the IgH template DNA was, the more active the transcription. It is suggested that the free energy of supercoiling promotes the formation of an open complex for initiation of transcription by the minimal set of transcription factors.

Cloning, Molecular↗
PubMed1993

Purification of the cellular C1 factor required for the stable recognition of the Oct-1 homeodomain by the herpes simplex virus alpha-trans-induction factor (VP16).

The assembly of specific multiprotein complexes on the herpes simplex virus alpha/IE (immediate early) enhancer elements requires the interactions of the Oct-1 POU homeodomain, the viral alpha TIF (alpha-trans-induction factor) (VP16), and at least one additional cellular factor, the C1 factor. The C1 factor interacts directly with alpha TIF, likely forming an intermediate protein complex that recognizes the Oct-1 homeodomain-DNA complex. The biochemical purification of the mammalian C1 factor suggests that it is composed of multiple subunits of related, but heterogeneous, polypeptides. The interaction of a subset of these polypeptides with alpha TIF is stimulated by post-translational modifications of the C1 proteins, suggesting that this factor may be a critical target for the regulation of the herpes simplex virus alpha/IE transcription.

Amino Acid Sequence↗
PubMed1993

High affinity DNA-binding Myc analogs: recognition by an alpha helix.

Myc and other basic-helix-loop-helix-leucine zipper (b-HLH-ZIP) proteins bind the sequence CACGTG. Exhaustive mutagenesis in the basic domain identified four amino acids critical for DNA binding with spacing suggestive of an alpha-helical face. Surprisingly, two highly conserved amino acids were nonessential for DNA binding. Circular dichroism demonstrated a DNA-induced alpha-helical transition. A series of analogs were constructed with multiple simultaneous alanine substitutions at nonessential positions and a critical lysine for arginine substitution. In this way 35-fold higher specific affinity for CACGTG was obtained as compared with the basic domain of c-Myc. These b-HLH-ZIP proteins appear to bind the same palindromic sequence and may compete for common sites in vivo. Additionally, a C-terminal basic region clamp motif was identified that was also identifiable in crystal structures from several different families of DNA-binding factors.

Alanine↗
PubMed1993

Acute and chronic effects of pancreatic glucagon on sugar transport across the brush-border and basolateral membranes of rat jejunal enterocytes.

Preparations of isolated rat jejunal enterocyte and brush-border and basolateral membrane vesicles have been used to study the effects of a 15 min exposure of upper and mid-villus enterocytes to pancreatic glucagon on the initial, unidirectional phlorhizin-sensitive (brush border) transport of galactose and phlorhizin-insensitive (basolateral) movement of the sugar. These acute effects of glucagon have been compared with responses following treatment of animals for 1 or 3 days with the hormone. Incubation of cells with glucagon significantly stimulated phlorhizin-sensitive uptake by 42 and 64% for upper and mid-villus cells, respectively. Glucagon, however, was without effect on phlorhizin-insensitive galactose uptake. This differential action of the hormone at the two cellular loci was confirmed by uptake data obtained using purified brush-border and basolateral membrane vesicles prepared from isolated cells. In contrast to the acute challenge with glucagon, treatment of animals for 3 days with the hormone significantly increased both phlorizin-sensitive (upper villus +31%, mid-villus +74%) and phlorizin-insensitive (upper villus +42%, mid-villus +53%) galactose uptake. Glucagon exposure of exposure of isolated cells from 3 days treated animals was without further effect on galactose uptake at the two membrane loci. These data represent the first evidence for a direct action of pancreatic glucagon on enterocyte sugar transport. Thus the hormone is likely to be important in the physiological control of sugar absorption in addition to its possible role in the modulation of transport during starvation and diabetes mellitus, conditions characterized by hyperglucanonaemia and enhanced intestinal sugar transport.

Animals↗