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At least 19 recordsLinked to original sources

Cloning of DNA corresponding to rare transcripts of rat brain: evidence of transcriptional and post-transcriptional control and of the existence of nonpolyadenylated transcripts.

To examine the expression of genes encoding rare transcripts in the rat brain, we have characterized genomic DNA clones corresponding to this class. In brain cells, as in all cell types, rare transcripts constitute the majority of different sequences transcribed. Moreover, when compared with other tissues or cultured cells, brain tissue may be expected to have an even larger set of rare transcripts, some of which could be restricted to subpopulations of neural cells. We have identified seven clones whose transcripts are nonabundant, averaging less than three copies per cell. Clone rg13 (rat genomic 13) RNA was detected only in the brain, whereas RNA of a second clone, rg40, was also detected in the brain and in a melanoma. Transcripts of rg13 were found in cerebellum, cerebral cortex, and regions underlying the cortex, whereas rg40 transcripts were not detected in the cerebellum. Transcripts of both rg13 and rg40 were found in pelleted polysomal RNA. RNA of another clone, rg34, was found in the brain, liver, and kidney but was found in pelleted polysomal RNA only in the brain, suggesting that its expression may be post-transcriptionally controlled. The remaining four clones represent rare transcripts that are common to the brain, liver, and kidney; rg18 RNA is restricted to the nucleus, whereas rg3, rg26, and rg36 transcripts are found in the cytoplasm of all three tissues. Transcripts of the brain-specific clone, rg13, and the commonly expressed clone, rg3, are nonpolyadenylated, presumably belonging to the high-complexity, nonpolyadenylated class of transcripts in the mammalian brain.

Animals↗

Transcription of a U6 small nuclear RNA gene in vitro. Transcription of a mouse U6 small nuclear RNA gene in vitro by RNA polymerase III is dependent on transcription factor(s) different from transcription factors IIIA, IIIB, and IIIC.

U6 small nuclear RNA (snRNA), an essential component of the eukaryotic spliceosomes, is unique in that it is synthesized by RNA polymerase III, while all other U-snRNAs are synthesized by RNA polymerase II. U6 genes are notable for functional upstream regulatory elements which resemble RNA polymerase II regulatory sequence motifs. In this study, the optimal conditions for transcription of the U6 snRNA gene in vitro were found to be similar to conditions optimal for transcription of 5S RNA genes. To purify the trans-acting factors necessary for the transcription of the U6 RNA gene, HeLa cell extracts were fractionated on a DEAE-Sephadex column, and three fractions, designated DE-50, DE-175, and DE-500, were obtained by stepwise elution with 50, 175, and 500 mM ammonium sulfate, respectively. DE-175 fraction transcribed tRNA and 5S RNA genes but not a mouse U6 RNA gene. Complementation of the DE-175 fraction with the DE-50 fraction resulted in the transcription of the U6 RNA gene. Experiments in which the transcription factor (TFIIIA) was selectively inactivated indicated that TFIIIA is not required for the transcription of the U6 RNA gene. These results show that the U6 snRNA gene, although transcribed by RNA polymerase III, differs from tRNA and 5S RNA genes in that factors other than TFIIIA, -IIIB, and -IIIC are required for U6 gene transcription in vitro.

Cloning, Molecular↗

Bacterial lipopolysaccharide stimulates the thyrotropin-dependent thyroglobulin gene expression at the transcriptional level by involving the transcription factors thyroid transcription factor-1 and paired box domain transcription factor 8.

The bacterial lipopolysaccharide (LPS) is a biological activator that induces expression of multiple genes in several cell types. LPS has been proposed as an etiopathogenic agent in autoimmune diseases. However, whether LPS affects the expression of autoantigens has not been explored. Thyroglobulin (TG) is a key protein in thyroid hormonogenesis and one of the major thyroid autoantigens. This study aimed to analyze the action of LPS on TG gene expression in Fisher rat thyroid cell line FRTL-5 thyroid cells. We demonstrate that LPS increases the TSH-induced TG protein and mRNA level. Evidence that the effect of LPS is exerted at the transcriptional level was obtained by transfecting the minimal TG promoter. The C element of the TG promoter, which contains sequences for paired box domain transcription factor 8 (Pax8) and thyroid transcription factor (TTF)-1 binding, is essential for full TG promoter expression under TSH stimulation. The transcriptional activity of a construct containing five tandem repeats of the C site is increased by LPS, indicating a possible involvement of the C site in the LPS-induced TG gene transcription. We demonstrate that the TG promoter mutated at the Pax8 or TTF-1 binding element in the C site does not respond to LPS. In band shift assays, binding of Pax8 and TTF-1 to the C site is increased by LPS. The Pax8 and TTF-1 mRNA and protein levels are augmented by LPS. The half-lives of TG, Pax8, and TTF-1 are increased in endotoxin-treated cells. Our results reveal the ability of LPS to stimulate the expression of TG, a finding of potential pathophysiological implication.

Animals↗

The CD3 delta gene encodes multiple transcripts regulated by transcriptional and post-transcriptional mechanisms.

CD3 is a multi-subunit complex of proteins noncovalently associated with the T cell receptor (TcR) for antigen. Considerable evidence indicates a role for CD3 molecules in the transduction of activation signals in T cells. The murine CD3 delta gene encodes a 0.7-kb transcript present in mature T cells. Here we report the characterization of several additional CD3 delta transcripts; two nuclear transcripts, 4-4.5 kb in size, and two predominamtly cytoplasmic transcripts of 1.5 kb and 2.5 kb. Both T lymphoma cell lines and normal thymocytes express the 1.5-kb and 2.5-kb CD3 delta transcripts. These cytoplasmic transcripts have long 3'-untranslated sequences which extend beyond the polyadenylation site of the predominant 0.7-kb transcript. The protein synthesis inhibitor cycloheximide (CHX) increases the expression of all three cytoplasmic CD3 delta transcripts, indicating that their level of expression may be regulated by a labile inhibitor protein(s). The CHX elicited increase in CD3 delta mRNA appears to result from post-transcriptional events since the rate of CD3 delta gene transcription remains constant. In contrast to CHX, the calcium ionophore A23187 increases the rate of CD3 delta gene transcription and, like CHX, also increases the level of cellular CD3 delta mRNA. The immunosuppressive agent cyclosporin A inhibits A23187-mediated stimulation of transcription, but has no effect on the CHX-mediated induction of CD3 delta mRNA. We conclude that both transcriptional and post-transcriptional mechanisms can regulate the amount of all three cytoplasmic CD3 delta transcripts.

Animals↗

Transcription factor B contacts promoter DNA near the transcription start site of the archaeal transcription initiation complex.

Transcription initiation in all three domains of life requires the assembly of large multiprotein complexes at DNA promoters before RNA polymerase (RNAP)-catalyzed transcript synthesis. Core RNAP subunits show homology among the three domains of life, and recent structural information supports this homology. General transcription factors are required for productive transcription initiation complex formation. The archaeal general transcription factors TATA-element-binding protein (TBP), which mediates promoter recognition, and transcription factor B (TFB), which mediates recruitment of RNAP, show extensive homology to eukaryal TBP and TFIIB. Crystallographic information is becoming available for fragments of transcription initiation complexes (e.g. RNAP, TBP-TFB-DNA, TBP-TFIIB-DNA), but understanding the molecular topography of complete initiation complexes still requires biochemical and biophysical characterization of protein-protein and protein-DNA interactions. In published work, systematic site-specific protein-DNA photocrosslinking has been used to define positions of RNAP subunits and general transcription factors in bacterial and eukaryal initiation complexes. In this work, we have used systematic site-specific protein-DNA photocrosslinking to define positions of RNAP subunits and general transcription factors in an archaeal initiation complex. Employing a set of 41 derivatized DNA fragments, each having a phenyl azide photoactivable crosslinking agent incorporated at a single, defined site within positions -40 to +1 of the gdh promoter of the hyperthermophilic marine archaea, Pyrococcus furiosus (Pf), we have determined the locations of PfRNAP subunits PfTBP and PfTFB relative to promoter DNA. The resulting topographical information supports the striking homology with the eukaryal initiation complex and permits one major new conclusion, which is that PfTFB interacts with promoter DNA not only in the TATA-element region but also in the transcription-bubble region, near the transcription start site. Comparison with crystallographic information implicates the PfTFB N-terminal domain in the interaction with the transcription-bubble region. The results are discussed in relation to the known effects of substitutions in the TFB and TFIIB N-terminal domains on transcription initiation and transcription start-site selection.

Archaeal Proteins↗

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

Transcription of a poxvirus early gene is regulated both by a short promoter element and by a transcriptional termination signal controlling transcriptional interference.

The promoter region of an early gene (38K gene) of cowpox virus has been characterized by deletion and linker scanning mutational analyses. Modified versions of this promoter region were placed into the genome of vaccinia virus, and their transcriptional efficiencies were assessed by quantifying RNAs transcribed from these sequences. These analyses showed that the sequences in the region between 33 and 4 base pairs upstream of the transcriptional start site affect the efficiency of transcription from this promoter. Linker scanning mutations in the -27 to -10 region inhibited transcription. This region contains the sequence 5'-GAAAATATATT-3', which is present in at least two other early genes in the same positions (-21 to -11) relative to the transcriptional start sites of these genes. Elements of this sequence are similarly positioned in the promoter regions of several other poxvirus genes, suggesting that this sequence represents a transcriptional control element of at least a subset of poxvirus genes. The -8 to -2 sequence (5'-TTTTTAT-3') contains a transcriptional termination signal. Mutation of this sequence had two separate effects: (i) it reduced the efficiency of transcription from the promoter by approximately 30%, and (ii) it prevented this sequence from terminating the transcription from upstream genes. When overlapping transcription from upstream genes was not prevented by a termination signal present either within the 38K promoter or upstream of the promoter, transcription from this promoter was reduced by about 30%. This indicates that transcriptional termination has a role in the regulation of viral gene expression by controlling transcriptional interference.

Amino Acid Sequence↗

Construction of a novel RNA-transcript-trimming plasmid which can be used both in vitro in place of run-off and (G)-free transcriptions and in vivo as multi-sequences transcription vectors.

We have constructed a new transcription system that allows trimming of both 5' and 3'-termini of any RNA transcripts by means of cis-acting ribozyme activities. The vector consists of a promoter, '5' Processing Ribozyme', any DNA template to be transcribed, and '3' Processing Ribozyme' sequences. When the vector possessing T7 promoter was tested in vitro, the transcription efficiency from the circular template was over ten-fold higher than using linearized template (run-off transcription). Further, since uniform RNAs with defined 5'- and 3'-ends can be produced, this strategy complements the conventional run-off transcription. Also the 5'-/3'-trimmed uniform RNA can function as a reporter in elucidating transcription factors and promoter regions in vitro, this strategy can replace the widely used (G)-free transcription (Sawadogo and Roeder (1985) Proc. Natl. Acad. Sci. USA 82, 4394-4398). With this strategy, in addition to the advantage that the template DNA need not be linearized prior to transcription, a cytidine-minus sequence is no longer necessary for quantitative analysis of transcription factors. Since any sequences including those of RNA virus can be inserted between the '5' Processing Ribozyme' and the '3' Processing Ribozyme' sequences, and the entire unit can be inserted into any genes under active transcription, this construct is useful like that of Dzianott and Bujarski ((1989) Proc. Natl. Acad. Sci. USA 86, 4823-4827) for RNA virologists because these strategies provide RNA transcripts without heterologous sequences which may greatly diminish infectivity. Moreover, since the construct can also be used in vivo, multi-transcripts such as trans-acting ribozymes targeted to various sites would be produced by concatenating the entire units in tandem.

Base Sequence↗

GreA-induced transcript cleavage in transcription complexes containing Escherichia coli RNA polymerase is controlled by multiple factors, including nascent transcript location and structure.

The Escherichia coli GreA and GreB proteins induce cleavage of 3' fragments from nascent transcripts in halted transcription complexes. We have overproduced and purified the GreA protein and tested how it affects initiation, pausing, and termination by E. coli RNA polymerase. Recombinant GreA induced cleavage of two to three nucleotide fragments in two promoter-proximal complexes, whereas an apparently endogenous cleavage removed a single larger fragment. Both types of cleavage stopped once the transcript was shortened to approximately 10 nucleotides. However, during initiation, GreA induced cleavage of transcripts as short as four nucleotides, inhibiting their release as abortive products and stimulating both productive initiation and "primer-shifting" at a weak promoter. GreA induced repetitive cleavage over a long distance in complexes containing a long G-less nascent transcript. However, reverse translocation was inhibited in transcription complexes that contained a G-rich, C-less nascent transcript. Substituting IMP for GMP in the transcript relieved inhibition. Finally, GreA had little effect on transcription through the his and trp leader pause sites or on termination at nine different p-independent terminators. We propose that transcript cleavage and reverse translocation are controlled in part by backsliding of the nascent transcript through an RNA-binding site.

Bacterial Proteins↗

Rice TATA binding protein interacts functionally with transcription factor IIB and the RF2a bZIP transcriptional activator in an enhanced plant in vitro transcription system.

TATA binding protein (TBP) and transcription factor IIB (TFIIB) are key factors for the assembly of eukaryotic transcription initiation complexes. We used a rice whole-cell extract in vitro transcription system to characterize the functional interactions of recombinant plant TBP and TFIIB. Bacterially expressed rice TBP (OsTBP2) bound to the TATA box of the rice pal gene encoding phenylalanine ammonia-lyase, caused DNA bending, and enhanced basal transcription from the pal promoter in a TATA box-dependent manner. Recombinant rice TFIIB (OsTFIIB) stimulated the DNA binding and bending activities of OsTBP2 and synergistically enhanced OsTBP2-mediated transcription from the pal promoter and the promoter of Rice tungro bacilliform virus but not from the barley pr1 promoter. We also demonstrate a physical interaction between OsTBP2 and RF2a, a rice bZIP transcription factor that bound to the box II cis element of the promoter of Rice tungro bacilliform virus, resulting in enhanced transcription from the viral promoter. Enhancement of rice whole-cell extracts with recombinant transcription factors thus provides a powerful tool for the in vitro determination of plant gene regulation mechanisms. We conclude that OsTBP2 undergoes promoter-specific functional interactions with both the basal transcription factor OsTFIIB and the accessory transcription factor RF2a.

Basic-Leucine Zipper Transcription Factors↗

The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis.

The mammalian ATF/CREB family of transcription factors represents a large group of basic region-leucine zipper (bZip) proteins which was originally defined in the late 1980s by their ability to bind to the consensus ATF/CRE site 'TGACGTCA'. Over the past decade, cDNA clones encoding identical or homologous proteins have been isolated by different laboratories and given different names. These proteins can be grouped into subgroups according to their amino acid similarity. In this review, we will briefly describe the classification of these proteins with a historical perspective of their nomenclature. We will then review three members of the ATF/CREB family of proteins: ATF3, ATF4 and ATF6. We will address four issues for each protein: (a) homologous proteins and alternative names, (b) dimer formation with other bZip proteins, (c) transcriptional activity, and (d) potential physiological functions. Although the name Activating Transcription Factor (ATF) implies that they are transcriptional activators, some of these proteins are transcriptional repressors. ATF3 homodimer is a transcriptional repressor and ATF4 has been reported to be either an activator or a repressor. We will review the reports on the transcriptional activities of ATF4, and propose potential explanations for the discrepancy. Although the physiological functions of these proteins are not well understood, some clues can be gained from studies with different approaches. When the data are available, we will address the following questions. (a) How is the expression (at the mRNA level or protein level) regulated? (b) How are the transcriptional activities regulated? (c) What are the interacting proteins (other than bZip partners)? (d) What are the consequences of ectopically expressing the gene (gain-of-function) or deleting the gene (loss-of-function)? Although answers to these questions are far from being complete, together they provide clues to the functions of these ATF proteins. Despite the diversity in the potential functions of these proteins, one common theme is their involvement in cellular responses to extracellular signals, indicating a role for these ATF proteins in homeostasis.

Activating Transcription Factor 2↗

Mapping regions of yeast transcription factor IIIA required for DNA binding, interaction with transcription factor IIIC, and transcription activity.

TFIIIA, a site-specific DNA-binding protein containing nine zinc finger motifs, is an RNA polymerase III transcription factor responsible for the assembly of a transcription complex on the 5 S RNA gene. We have analyzed the effect of deleting various regions of yeast TFIIIA on its ability to bind to the internal control region of the yeast 5 S RNA gene, to recruit TFIIIC into the TFIIIA.DNA complex and to promote in vitro transcription of the 5 S RNA gene. A truncated polypeptide containing only the three amino-terminal zinc fingers retained the ability to bind specifically to the internal control region of the 5 S RNA gene. The TFIIIA.DNA complex formed with this three zinc finger module was able to recruit TFIIIC but could not promote transcription. An 81-amino acid domain, which interrupts the repeating zinc finger motifs between fingers 8 and 9, was found to be essential for the transcription activity of the protein. Removal of the carboxyl-terminal region of yeast TFIIIA that extends beyond the ninth zinc finger motif had no effect on the in vitro activities of the protein. Extending the carboxyl-terminal deletion to include the ninth zinc finger led to reduced transcription activity. We speculate that the ninth zinc finger serves to anchor the carboxyl-terminal portion of TFIIIA on the 5 S RNA gene, correctly orienting the 81-amino acid domain to serve its role in promoting transcription. Removal of the region of the protein amino-terminal to the first zinc finger motif was without effect. Extending the amino-terminal deletion to include the first zinc finger appeared to decrease the affinity of TFIIIA for DNA without affecting the specificity of the protein-DNA interaction. The resultant TFIIIA.DNA complex was defective in recruiting TFIIIC and did not support transcription.

Amino Acid Sequence↗

Splicing factors associate with nuclear HCMV-IE transcripts after transcriptional activation of the gene, but dissociate upon transcription inhibition: evidence for a dynamic organization of splicing factors.

Before being transported to the cytoplasm, intron-containing pre-mRNAs have to be spliced somewhere in the cell nucleus. Efficient splicing requires an ordered assembly of splicing factors onto the pre-mRNAs. To accomplish this, intron containing genes may be preferentially localized at nuclear sites enriched for splicing factors or alternatively, splicing factors may circulate throughout the nucleus and have the ability to associate with randomly positioned nascent transcripts. Combined detection of HCMV-IE mRNA/DNA and splicing factors in rat 9G cells that can be induced for IE gene expression shows that IE genes are not associated with speckled regions enriched for splicing factors when transcriptionally inactive, but 'attract' splicing factors when transcriptionally activated. This process proved reversible after transcription inhibition. IE transcripts appeared to be retained near the transcription site in track-like domains by splicing factors associated with them until splicing has been completed. Double-hybridization experiments revealed that a substantial part of the accumulated transcripts contain a poly(A) tail suggesting that most, if not all, IE transcripts are polyadenylated at the site of transcription. These results indicate that RNA processing may occur independent of the position of the gene in the cell nucleus relative to speckle domains.

Animals↗

Regulation of metallothionein transcription by the metal-responsive transcription factor MTF-1: identification of signal transduction cascades that control metal-inducible transcription.

Every living organism must detoxify nonessential metals and carefully control the intracellular concentration of essential metals. Metallothioneins, which are small, cysteine-rich, metal-binding proteins, play an important role in these processes. In addition, the transcription of their cognate genes is activated in response to metal exposure. The zinc finger transcription factor MTF-1 plays a central role in the metal-inducible transcriptional activation of metallothionein and other genes involved in metal homeostasis and cellular stress response. Here we report that the phosphorylation of MTF-1 plays a critical role in its activation by zinc and cadmium. Inhibitor studies indicate that multiple kinases and signal transduction cascades, including those mediated by protein kinase C, tyrosine kinase, and casein kinase II, are essential for zinc- and cadmium-inducible transcriptional activation. In addition, calcium signaling is also involved in regulating metal-activated transcription. In contrast, cAMP-dependent protein kinase may not be directly involved in the metal response. Contrary to what has been reported for other transcription factors, inhibition of transcriptional activation does not impair the binding of MTF-1 to DNA, suggesting that phosphorylation is not regulating DNA binding. Elevated phosphorylation of MTF-1 is observed under condition of protein kinase C inhibition, suggesting that specific dephosphorylation of this transcription factor contributes to its activation.

Animals↗

Two transcription factors related with the eucaryal transcription factors TATA-binding protein and transcription factor IIB direct promoter recognition by an archaeal RNA polymerase.

We reported previously that cell-free transcription in the Archaea Methanococcus and Pyrococcus depends upon two archaeal transcription factors, archaeal transcription factor A (aTFA) and archaeal transcription factor B (aTFB). In the genome of Pyrococcus genes encoding putative homologues of eucaryal transcription factors TATA-binding protein (TBP) and TFIIB have been detected. Here, we report that Escherichia coli synthesized Pyrococcus homologues of TBP and TFIIB are able to replace endogenous aTFB and aTFA in cell-free transcription reactions. Antibodies raised against archaeal TBP and TFIIB bind to polypeptides of identical molecular mass in the aTFB and aTFA fraction. These data identify aTFB as archaeal TBP and aTFA as the archaeal homologue of TFIIB. At the Pyrococcus glutamate dehydrogenase (gdh) promoter these two bacterially produced transcription factors and endogenous RNA polymerase are sufficient to direct accurate and active initiation of transcription. DNase I protection experiments revealed Pyrococcus-TBP producing a characteristic footprint between position -20 and -34 centered around the TATA box of gdh promoter. Pyrococcus-TFIIB did not bind to the TATA box but bound cooperatively with Pyrococcus-TBP generating an extended DNase I footprinting pattern ranging from position -19 to -42. These data suggest that the Pyrococcus homologue of TFIIB associates with the TBP-promoter binary complex as its eucaryal counterpart, but in contrast to eucaryal TFIIB, it causes an extension of the protection to the region upstream of the TATA box.

Archaea↗

Association of COOH-terminal-binding protein (CtBP) and MEF2-interacting transcription repressor (MITR) contributes to transcriptional repression of the MEF2 transcription factor.

The class II histone deacetylases (HDACs) 4, 5, and 7 share a common structural organization, with a carboxyl-terminal catalytic domain and an amino-terminal extension that mediates interactions with members of the myocyte enhancer factor-2 (MEF2) family of transcription factors. Association of these HDACs with MEF2 factors represses transcription of MEF2 target genes. MEF2-interacting transcription repressor (MITR) shares homology with the amino-terminal extensions of class II HDACs and also acts as a transcriptional repressor, but lacks a histone deacetylase catalytic domain. This suggests that MITR represses transcription by recruiting other corepressors. We show that the amino-terminal regions of MITR and class II HDACs interact with the transcriptional corepressor, COOH-terminal-binding protein (CtBP), through a CtBP-binding motif (P-X-D-L-R) conserved in MITR and HDACs 4, 5, and 7. Mutation of this sequence in MITR abolishes interaction with CtBP and impairs, but does not eliminate, the ability of MITR to inhibit MEF2-dependent transcription. The residual repressive activity of MITR mutants that fail to bind CtBP can be attributed to association with other HDAC family members. These findings reveal CtBP-dependent and -independent mechanisms for transcriptional repression by MITR and show that MITR represses MEF2 activity through recruitment of multicomponent corepressor complexes that include CtBP and HDACs.

Alcohol Oxidoreductases↗

Effects of endogenous DNA base lesions on transcription elongation by mammalian RNA polymerase II. Implications for transcription-coupled DNA repair and transcriptional mutagenesis.

The blockage of transcription elongation by RNA polymerase II (pol II) is thought to be a trigger for transcription-coupled repair in the pathway of nucleotide excision repair. Purified pol II and oligo(dC)-tailed templates containing a single non-bulky DNA lesion on the transcribed strand such as an apurinic/apyrimidinic (AP) site, uracil, or 8-oxoguanine (8-oxoG) were used for transcription elongation assays. In this system pol II could bypass both the AP site and uracil without pausing and insert cytosine opposite the AP site and either guanine or adenine opposite to uracil. Thus, the AP site on the DNA templates could lead to correct transcription only if depurination at guanine occurred, whereas uracil generated either the correct transcriptional product or an incorrect one with a G:C to A:T transition. In the case of 8-oxoG, pol II stalled at the lesion, but sometimes bypassed it and inserted a cytosine residue or the incorrect adenine residue leading to a G:C to T:A transversion. These findings indicate that 8-oxoG lesions caused a blockage of transcription elongation and/or the misincorporation of a ribonucleotide by pol II, implying the initiation of transcription-coupled repair of 8-oxoG and/or transcriptional mutagenesis.

Adenine↗

Cell-free transcription at 95 degrees: thermostability of transcriptional components and DNA topology requirements of Pyrococcus transcription.

Cell-free transcription of archaeal promoters is mediated by two archaeal transcription factors, aTBP and TFB, which are orthologues of the eukaryotic transcription factors TBP and TFIIB. Using the cell-free transcription system described for the hyperthermophilic Archaeon Pyrococcus furiosus by Hethke et al., the temperature limits and template topology requirements of archaeal transcription were investigated. aTBP activity was not affected after incubation for 1 hr at 100 degrees. In contrast, the half-life of RNA polymerase activity was 23 min and that of TFB activity was 3 min. The half-life of a 328-nt RNA product was 10 min at 100 degrees. Best stability of RNA was observed at pH 6, at 400 mm K-glutamate in the absence of Mg(2+) ions. Physiological concentrations of K-glutamate were found to stabilize protein components in addition, indicating that salt is an important extrinsic factor contributing to thermostability. Both RNA and proteins were stabilized by the osmolyte betaine at a concentration of 1 m. The highest activity for RNA synthesis at 95 degrees was obtained in the presence of 1 m betaine and 400 mm K-glutamate. Positively supercoiled DNA, which was found to exist in Pyrococcus cells, can be transcribed in vitro both at 70 degrees and 90 degrees. However, negatively supercoiled DNA was the preferred template at all temperatures tested. Analyses of transcripts from plasmid topoisomers harboring the glutamate dehydrogenase promoter and of transcription reactions conducted in the presence of reverse gyrase indicate that positive supercoiling of DNA inhibits transcription from this promoter.

Archaeal Proteins↗