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C M Kane

Publications and source records attributed to C M Kane.

At least 37 records · Page 2Linked to original sources
PubMed1996

Purification and characterization of an RNA polymerase II phosphatase from yeast.

RNA polymerase (RNAP) II is subject to extensive phosphorylation on the heptapeptide repeats of the C-terminal domain (CTD) of the largest subunit. An activity that is required for the dephosphorylation of yeast RNAP II in vitro has been purified from a yeast whole cell extract by >30,000-fold. The yeast CTD phosphatase activity copurified with two bands with apparent molecular masses of 100 and 103 kDa. The properties of the yeast CTD phosphatase are similar to those of a previously characterized CTD phosphatase from HeLa cells. These properties include stimulation by the general transcription factor IIF (TFIIF), competitive inhibition by RNAP II, magnesium dependence, and resistance to okadaic acid. Both the HeLa and yeast CTD phosphatases are highly specific for their cognate polymerases. Neither phosphatase functions upon the polymerase molecule from the other species, even though the heptapeptide repeats of the CTDs in yeast RNAP II and mammalian RNAP II are essentially identical. The activity of the highly purified CTD phosphatase is stimulated >300-fold by a partially purified fraction of TFIIF. Recombinant TFIIF did not substitute for the TFIIF fraction, indicating that an additional factor present in the TFIIF fraction is required for CTD phosphatase activity. These results show that yeast contains a CTD phosphatase activity similar to that of mammalian cells that is likely composed of at least two components, one of which is 100 and/or 103 kDa.

Chromatography, Ion Exchange↗
PubMed1996

Isolation and characterization of the Schizosaccharomyces pombe gene encoding transcript elongation factor TFIIS.

A gene designated tfs1 has been isolated from Schizosaccharomyces pombe based on its similarity to genes encoding transcription elongation factor TFIIS. The nucleotide sequence of the tfs1 gene predicts a polypeptide with similarity to mammalian. Drosophila and Saccharomyces cerevisiae TFIIS. A haploid Sz. pombe strain with tfs1 deleted from the genome is viable. Thus, tfs1 is not essential for viability. However, deletion of tfs1 results in slow growth and increased sensitivity to the drug 6-azauracil, a phenotype similar to that of a S. cerevisiae strain deleted for the gene encoding TFIIS. The DNA sequence of tfs1 has been deposited in GenBank under Accession Number U20526.

Base Sequence↗
PubMed1995

Alanine-scanning mutagenesis of human transcript elongation factor TFIIS.

TFIIS is a transcription elongation factor that binds to RNA polymerase II and allows it to transcribe through a variety of transcriptional blockages by inducing cleavage near the 3' end of the nascent transcript. Although this cleavage reaction plays a key role in the process of reactivation of transcription by TFIIS, the exact mechanism by which TFIIS promotes readthrough by RNA polymerase II is not completely understood. We therefore undertook a systematic mutagenesis of the C-terminal half of TFIIS (delta TFIIS) to evaluate the contribution of charged residues in this region to induce transcript cleavage and promote readthrough in vitro. Twenty-two delta TFIIS alanine-scanning mutants were constructed by substitution of alanine for each amino acid in clusters of charged residues in the C-terminal half of HeLa TFIIS. The ability to induce transcript cleavage and readthrough of these mutants was tested in vitro using RNA polymerase II ternary elongation complexes arrested at a block to elongation. This alanine-scanning mutagenesis analysis allowed the identification of regions or residues important for the activity of TFIIS. Many of the mutants were reduced alike in both cleavage and readthrough activities. However, in several cases there was no simple correlation between these activity reductions.

Alanine↗
PubMed1994

Cleavage of the nascent transcript induced by TFIIS is insufficient to promote read-through of intrinsic blocks to elongation by RNA polymerase II.

RNA polymerases encounter a variety of types of blocks to elongation during transcription in eukaryotic cells. At least one protein, TFIIS, can promote read-through of many types of blocks to elongation by RNA polymerase II, and this protein stimulates cleavage of the nascent transcript in stalled elongation complexes as a prelude to read-through. The C-terminal half of the TFIIS protein is sufficient for stimulating the cleavage and read-through reactions in vitro. To study how TFIIS changes the response of RNA polymerase II elongation complexes to such blocks, targeted amino acids in the C terminus of HeLa TFIIS were mutated to alanines. Two mutant TFIIS proteins as well as the unmutated C-terminal half of the TFIIS protein were purified following overexpression in Escherichia coli. Each protein was examined for read-through activity and ability to stimulate transcript cleavage in ternary elongation complexes. Mutant TFIIS5 (E174A, E175A) was reduced in read-through and cleavage activities relative to the unmutated, truncated TFIIS (delta TFIIS). Mutant TFIIS7 (K187A, K189A) was able to stimulate cleavage nearly at the rate and to the extent of the TFIIS5 mutant. In contrast to what was observed with TFIIS5, no detectable read-through was observed in the presence of the TFIIS7 mutant during the course of the reaction. Thus, there is no simple, direct correlation between the ability of TFIIS to promote cleavage and its ability to promote read-through by RNA polymerase II. These results suggest that although TFIIS is necessary to mediate the cleavage reaction that precedes the read-through event, the cleavage event itself is not sufficient to allow read-through by RNA polymerase II.

Amino Acid Sequence↗
PubMed1994

Purified yeast RNA polymerase II reads through intrinsic blocks to elongation in response to the yeast TFIIS analogue, P37.

Saccharomyces cerevisiae has a TFIIS-related transcription elongation factor, originally called P37 (Sawadogo, M., Sentenac, A., and Fromageot, P. (1979) J. Biol. Chem. 255, 12-15; Nakanishi, T., Nakano, A., Nomura, K., Sekimizu, K., and Natori, S. (1992) J. Biol. Chem. 267, 13200-13204), which binds directly to RNA polymerase II and stimulates read-through of intrinsic blocks to elongation. To elucidate functional features of this protein:protein interaction, we tested the ability of several forms of RNA polymerase II to respond to either full-length or an amino-terminal truncation of TFIIS. The variants of the polymerase differed in the structure of the carboxyl-terminal domain of the largest subunit or lacked two of the smaller subunits. No differences in ability to recognize intrinsic blocks to elongation or to read through them in response to either form of TFIIS were detected among these variants. Furthermore, ternary complexes containing each variant form of RNA polymerase cleave the 3' end of the nascent transcripts in response to TFIIS, a reaction previously reported for mammalian and Drosophila TFIIS (Kassavetis, G. A., and Geiduschek, E. P. (1993) Science 259, 944-945) and likely to be important in TFIIS function. Thus the carboxyl-terminal domain of the largest subunit and subunits four and seven of the polymerase, required in vivo, are not required in vitro for recognition of intrinsic blocks to elongation, read-through in response to TFIIS, or TFIIS-stimulated cleavage of the nascent transcript.

Base Sequence↗
PubMed1993

Contacts between mammalian RNA polymerase II and the template DNA in a ternary elongation complex.

Elongation complexes of RNA polymerase II, RNA-DNA-enzyme ternary complexes, are intermediates in the synthesis of all eukaryotic mRNAs and are potential regulatory targets for factors controlling RNA chain elongation and termination. Analysis of such complexes can provide information concerning the structure of the catalytic core of the RNA polymerase and its interactions with the DNA template and RNA transcript. Knowledge of the structure of such complexes is essential in understanding the catalytic and regulatory properties of RNA polymerase. We have prepared and isolated complexes of purified RNA polymerase II halted at defined positions along a DNA template, and we have used deoxyribonuclease I (DNAse I) to map the interactions of the polymerase with the DNA template. DNAse I footprints of three specific ternary complexes reveal that the enzyme-template interactions of individual elongation complexes are not identical. The size of the protected region is distinct for each complex and varies from 48 to 55 bp between different complexes. Additionally, the positioning of the protected region relative to the active site varies in different complexes. Our results suggest that RNA polymerase II is a dynamic molecule and undergoes continual conformational transitions during elongation. These transitions are likely to be important in the processes of transcript elongation and termination and their regulation.

Animals↗
PubMed1993

TGF-beta 2 gene and protein expression in maternal and fetal tissues at various stages of murine development.

The transforming growth factor beta family of peptides have diverse actions on the reproductive tracts of primates and rodents. In this study we report the expression of high levels of mRNA of one member of this superfamily, TGF-beta 2, in the pregnant mouse uterus. Using Northern blot analysis and in situ hybridization techniques, we have examined the pattern of expression of TGF-beta 1, TGF-beta 2 and colony-stimulating factor (CSF-1) in mouse maternal and fetal tissue at specific days of gestation. We report here that TGF-beta 2 is synthesized primarily in maternal decidual and uterine epithelial tissues. We observed a shift in the major site of synthesis from decidua to uterus between days 8.5 and 10.5 of gestation. These data demonstrate that the expression of TGF-beta 2 is differentially regulated in the decidua and uterine epithelial cells at various times during gestation. Small amounts of TGF-beta 2 mRNAs were detected in the fetus, and none was detected in placenta, yolk sac, or amniotic membrane. The uterus is likely the major site of synthesis of the TGF-beta 2 found in mouse amniotic fluid. TGF-beta 1 mRNAs are expressed in the uterus at markedly lower levels when compared to TGF-beta 2 mRNAs in both the decidua and uterus. Our results suggest that there is a unique regulation of TGF-beta 2 during pregnancy which may depend on pregnancy hormone(s) and differentiates it from the other mammalian isoforms of the TGF-beta s. TGF-beta 2 may play an important, albeit unknown, role at the maternal/fetal interface.

Amniotic Fluid↗
PubMed1993

Expression of the mouse Ren-1 gene in the coagulating gland: localization and regulation.

The components of the renin-angiotensin system have been detected in various reproductive tissues of mammals including the testis, epididymis, ovary, and placenta. Using Northern blot and in situ hybridization analyses, we detected abundant levels of renin mRNA in the coagulating gland (anterior prostate) of mice with one (Ren-1) but not two (Ren-1/Ren-2) copies of the renin gene. In contrast to mice, the single renin gene of the rat (R. norvegicus) was silent in the coagulating gland. The results of this survey suggest that the ability of the renin gene to express in the coagulating gland was acquired during the speciation of mice and subsequently lost as a result of the duplication event at the renin locus. In the coagulating gland, we found that renin mRNA transcripts initiated at a series of upstream start sites, some of which map within a 0.5-kb transposable-like element previously identified in the promoter region of the mouse, but not the rat, renin gene. Furthermore, renin gene expression in the coagulating gland was positively regulated by testosterone. The coagulating gland thus represents a male reproductive tissue that demonstrates high-level, species-specific, and differential expression of renin mRNA.

Animals↗
PubMed1992

Characterization of a HeLa cDNA clone encoding the human SII protein, an elongation factor for RNA polymerase II.

We present the cloning and sequence characterization of a HeLa cDNA encoding the SII transcription elongation factor. This cDNA clone is distinct from those previously isolated from a human kidney cDNA library [Yoo et al., Nucleic Acids Res. 19 (1991) 1073-1079]. Southern analysis suggests that more than one gene may exist for SII in the human genome. A comparison of deduced amino acid sequences for SII-related proteins from a variety of eukaryotes demonstrates very high similarity, especially within the C-terminal domain.

Amino Acid Sequence↗
PubMed1992

Regulated tissue- and cell-specific expression of the human renin gene in transgenic mice.

Transgenic mice containing the human renin gene were constructed with the aim of examining the tissue- and cell-specific expression of human renin. The human renin transgene used consisted of a genomic sequence extending approximately 900 bp upstream and 400 bp downstream of the coding region and included all exon and intron sequences. Two assays were developed to differentiate human renin transcripts from endogenous mouse renin transcripts at the whole-tissue level. High level human renin expression was evident in the kidney, adrenal gland, ovary, testis, lung, and adipose tissue of all four transgenic lines examined. Human renin mRNA could also be detected at lower levels in the submandibular gland and heart of two different individual lines. No expression was evident in the liver or brain of any line tested. In situ hybridization revealed the human renin mRNA to be localized and exquisitely restricted to renal juxtaglomerular cells. Treatment of transgenic mice with captopril resulted in an increase in the accumulation of renal renin mRNAs derived from both the mouse and human renin genes. Plasma renin activity assays using synthetic human renin substrate clearly demonstrated the elaboration of active human renin into the systemic circulation of transgenic mice. These data strongly suggest that the human renin transgene exhibits both tissue- and cell-specific expression in transgenic mice. Its expression is entrained to the same regulatory signals as the endogenous renin gene in kidney, and active human renin is released into the plasma of the transgenic mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Footprinting analysis of mammalian RNA polymerase II along its transcript: an alternative view of transcription elongation.

Ternary complexes of RNA polymerase II, bearing the nascent RNA transcript, are intermediates in the synthesis of all eukaryotic mRNAs and are implicated as regulatory targets of factors that control RNA chain elongation and termination. Information as to the structure of such complexes is essential in understanding the catalytic and regulatory properties of the RNA polymerase. We have prepared complexes of purified RNA polymerase II halted at defined positions along a DNA template and used RNase footprinting to map interactions of the polymerase with the nascent RNA. Unexpectedly, the transcript is sensitive to cleavage by RNases A and T1 at positions as close as 3 nucleotides from the 3'-terminal growing point. Ternary complexes in which the transcript has been cleaved to give a short fragment can retain that fragment and remain active and able to continue elongation. Since DNA.RNA hybrid structures are completely resistant to cleavage under our reaction conditions, the results suggest that any DNA.RNA hybrid intermediate can extend for no more than 3 base pairs, in dramatic contrast to recent models for transcription elongation. At lower RNase concentrations, the transcript is protected from cleavage out to about 24 nucleotides from the 3' terminus. We interpret this partial protection as due to the presence of an RNA binding site on the polymerase that binds the nascent transcript during elongation, a model proposed earlier by several workers in preference to the hybrid model. The properties of this RNA binding site are likely to play a central role in the process of transcription elongation and termination and in their regulation.

Animals↗
PubMed1991

Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro.

RNA polymerase II lacking the fourth and seventh largest subunits (pol II delta 4/7) was purified from Saccharomyces cerevisiae strain rpb-4, in which the gene for the fourth largest subunit is deleted. pol II delta 4/7 was indistinguishable from wild-type pol II (holoenzyme) in promoter-independent initiation/chain elongation activity (400-800 nmol of nucleotide incorporated/10 min/mg of protein at 22 degrees C), in rate of chain elongation (20-25 nucleotides/s), and in the recognition of pause sites in the DNA template. In contrast to pol II holoenzyme, pol II delta 4/7 was inactive in promoter-directed initiation of transcription in vitro. The addition of an equimolar complex of the fourth and seventh largest subunits, purified from pol II holoenzyme by ion-exchange chromatography in the presence of urea, restored promoter-directed initiation activity to pol II delta 4/7. The transcriptional activator protein Gal4-VP16 could also elicit promoter-directed initiation by pol II delta 4/7 from a promoter with a Gal4 binding site. Complementation was observed between extracts of strain rpb-4, lacking the fourth largest subunit, and strain Y260-1, with a defect in the largest subunit. These extracts were individually inactive, but a mixture would support promoter-directed initiation. The fourth and seventh largest subunits may, therefore, shuttle between polymerase molecules.

Cell Nucleus↗
PubMed1991

RNA polymerase: regulation of transcript elongation and termination.

Expanded interest in studying the mechanisms of elongation and termination during transcription has come as a result of several recent findings that highlight the importance of the regulation of these processes in human health. Several cellular proto-oncogenes contain regulated blocks to elongation (1), and the human immunodeficiency viruses also control gene expression in part by regulating the efficiency of elongation in response to the trans-activating protein, TAT (2). This review considers these recent findings and compares potential mechanisms of regulation used by prokaryotic and eukaryotic RNA polymerases during elongation and termination. In all these systems, many of the detailed mechanisms of transcription elongation and termination are still to be defined; however, we have tried to group examples that may share some common regulatory elements into simplified categories.

Animals↗
PubMed1990

Purified elongation factor SII is sufficient to promote read-through by purified RNA polymerase II at specific termination sites in the human histone H3.3 gene.

Purified RNA polymerase II terminates transcription in vitro at sites within genes which also block transcript elongation in vivo. Studies on a termination site within the first intron of the human histone H3.3 gene have shown that transcription elongation factor SII can promote read-through at this site when the polymerase initiates transcription from a promoter in the presence of the accessory initiation factors. Using 3'-extended templates to direct specific initiation by purified RNA polymerase II, we show here that purified SII is sufficient to effect read-through of this terminator by the purified polymerase alone. Thus, the interaction of purified SII with an elongation complex containing only the polymerase, the template, and the nascent transcript can change the termination properties of RNA polymerase II and can effect read-through of a region that blocks elongation in the cell.

Animals↗
PubMed1990

Analysis of the signals for transcription termination by purified RNA polymerase II.

Eukaryotic RNA polymerase II recognizes certain DNA sequences as effective signals for transcription termination in vitro. Previously, we have shown that such termination occurs within T-rich sequences; however, not all T runs stop the enzyme nor is the efficiency of termination correlated with the length of the T run. Here we have investigated the sequence elements that signal transcription termination by purified RNA polymerase II. We have examined terminators located within introns of the human histone H3.3 gene and the human c-myc gene. Deletion analysis of the H3.3 termination region indicates that the sequences between -6 and +24 relative to the strongest termination site are sufficient to cause transcription termination. The minimal termination signal at this site has been localized to the sequence TTTTTTTC-CCTTTTTT in the nontranscribed strand. A similar but nonidentical sequence has been defined for the c-myc termination site. Since RNA polymerase II terminates transcription only within the first run of T residues in these sequences, at least part of the termination signal lies in downstream nontranscribed DNA sequences. Restriction fragment mobility analysis indicates that the H3.3 termination region contains a bend in the DNA helix. Oligonucleotides containing the minimal termination signals also cause restriction fragments to migrate with anomalous mobility. A region of the SV40 genome containing a previously characterized bend also causes RNA polymerase II to terminate transcription. We suggest that a structural element causing a bend in the DNA helix may be part of the signal for transcription termination by purified RNA polymerase II.

Animals↗
PubMed1989

Transcription elongation factor SII (TFIIS) enables RNA polymerase II to elongate through a block to transcription in a human gene in vitro.

Elongation and termination by RNA polymerase II are important regulatory steps for eukaryotic gene expression. We have previously studied the transcription of linear DNA templates where specific initiation of transcription by highly purified RNA polymerase II can be achieved in the absence of promoters and promoter-specific factors. Using these templates we have shown that a human histone gene, H3.3, contains sequences (intrinsic terminators) within which purified RNA polymerase II will efficiently terminate transcription (Reines, D., Wells, D., Chamberlin, M.J., and Kane, C. M. (1987) J. Mol. Biol. 196, 299-312). Curiously, these signals were found within an intron, 3'-untranslated, and protein-encoding regions of the gene suggesting that they might act to attenuate transcription of H3.3 in vivo. Here we show that intrinsic terminator sequences from an H3.3 gene intron also block in vitro transcript elongation by RNA polymerase II when the enzyme has initiated transcription from a promoter using highly purified transcription initiation factors. However, under the conditions used for promoter-specific transcription there is little transcript release. Instead the polymerase can pause at these sites for periods exceeding 60 min. We have identified and partially purified an activity from HeLa cells that causes the transcription complex to read through this block to transcription elongation. This readthrough activity fractionates with a previously characterized elongation factor (SII) over three chromatographic columns. A homogeneous preparation of calf thymus SII can also provide this activity in trans. This factor may facilitate passage of the RNA polymerase II transcription complex through such intragenic sites in cellular genes in vivo.

Animals↗
PubMed1988

Renaturase and ribonuclease H: a novel mechanism that influences transcript displacement by RNA polymerase II in vitro.

I have previously reported an activity in HeLa cells which facilitates transcript displacement by purified mammalian RNA polymerase II in vitro. I have shown that this activity copurifies with one of two separable ribonuclease (RNase) H activities in HeLa cells. The RNase H activity in question has characteristics similar to those reported for RNase H2b from calf thymus. RNase H proteins purified from several other sources including Escherichia coli also show renaturase activity. When the renaturase/RNase H protein is present during transcription by purified RNA polymerase II, transcripts are truncated close to the 5' end, and the remainder of the transcript is displaced normally from its template by the polymerase. Since RNA polymerase II dependent transcripts in vivo normally require the presence of the 5'-triphosphate terminus for capping, the in vivo significance of RNase H as a renaturase factor is presently not understood. However, the in vitro action of renaturase/RNase H suggests that the mechanism of this reaction may involve R-loop displacement after formation of a short single-stranded region of DNA on the template strand following hydrolysis of a hybrid transcript oligonucleotide by RNase H.

Animals↗