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At least 55 records · Page 3Linked to original sources
PubMed1991

Down-regulation of lck mRNA by T cell activation involves transcriptional and post-transcriptional mechanisms.

The p56lck tyrosine kinase is most likely to be involved in signal transduction of T lymphocyte activation. After full activation through the TcR/CD3 complex lck mRNA is transiently down-modulated. This down-modulation was due to an early decrease of both transcription and stability of the lck mRNA. To study the involvement of transcriptional and post-transcriptional factors in this regulations, we have analysed the effect of cycloheximide, a protein synthesis inhibitor, on the steady-state of the lck mRNA. Cycloheximide superinduced lck mRNA by increasing its stability, although cycloheximide concomitantly decreased lck transcription. This suggests that the constitutive level of lck mRNA observed prior to activation is controlled by transcriptional activator(s) and post-transcriptional destabilizing factor(s). Second, lck mRNA down-modulation observed after full activation was inhibited by cycloheximide. It increased lck mRNA stability whereas lck transcription remained low. Therefore, full activation might increase the synthesis and/or activity of destabilizing factor(s). Cyclosporin A also inhibited the down-modulation of lck mRNA by increasing its transcription with no effect on its stability. Since, lck mRNA down-modulation was always associated with lymphokine mRNA induction, and since CsA blocks both lymphokine transcription and lck decrease of transcription, this indicates that these genes might share common regulatory pathways leading to their inverse transcriptional regulation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

Transcriptional and post-transcriptional regulation of L-type pyruvate kinase gene expression in rat liver.

The effects of starvation, refeeding a diet high in carbohydrate, administration of glucagon and cyclic AMP, thyroidectomy, and adrenalectomy on transcription of the gene for liver L-type pyruvate kinase and on the accumulation of cytoplasmic mRNA for L-type pyruvate kinase were investigated in rat. Transcription of the gene was undetectable in either fasted or protein-fed rats. Refeeding fasted rats a carbohydrate-rich diet stimulated an increase in L-type pyruvate kinase mRNA, preceded by an increase in the gene transcription. Transcription was maximal at 12 h of refeeding, decreasing to 10% of maximum at 72 h. The level of L-type pyruvate kinase mRNA remained constant at 50% of maximum for at least 120 h. Neither thyroidectomy nor adrenalectomy affected gene transcription in fasted rats refed the carbohydrate-rich diet, despite a decrease in mRNA abundance to 40 and 20%, respectively, of controls fed a normal diet. Glucagon or cyclic AMP totally blocked the increase in transcription of the L-type pyruvate kinase gene caused by feeding a carbohydrate-rich diet to previously fasted rats. Nevertheless, the level of L-type pyruvate kinase mRNA remained high for 3 h after glucagon administration. After 3 h, the mRNA decreased rapidly with a half-life less than 1 h. Thus, expression of the gene for L-type pyruvate kinase is regulated at both transcriptional and post-transcriptional levels. The transcription is regulated by two major effectors, one positive, namely carbohydrates, and one negative, namely glucagon (via cyclic AMP). Both agents probably act at the level of the mRNA stability as well. Glucocorticoids and thyroid hormones do not regulate transcription of the gene for L-type pyruvate kinase but do appear to be required for a normal accumulation of the transcripts in the cytoplasm.

Adrenalectomy↗
PubMed1992

Adenovirus VARNA1 gene B block promoter element sequences required for transcription and for interaction with transcription factors.

We constructed mutants with a deletion of either half of the 18 base-pair B block palindrome in the VARNA1 gene, mutants with different intra-palindromic spacings, a complete set of mutants with single base substitutions, and mutants with double and triple base substitutions in the palindrome. The transcription efficiencies of these mutants were determined in human KB cell-free cytoplasmic S100 extracts. The relative competing strength of each mutant, as determined by a sequential competition experiment, was used to assess each mutant's ability to sequester factors into formation of a stable preinitiation complex. The ability of each mutant to assemble transcriptionally active preinitiation complexes was also determined by direct transcription of the isolated complexes. Finally, the ability of each mutant to interact with the transcription factor(s) TFIIIC and form a distinct gel-resolved complex was also determined. From the results of the above assays, we concluded that the two seemingly identical halves of the palindrome did not contribute equally to transcription, or to assembly of the functional preinitiation complex, nor to interaction with TFIIIC. The anterior half (B1) of the B block palindrome, which is proximal to the A block promoter element, played a stronger role in transcription and in assembly of the functional preinitiation complex than the posterior half (B2) of the palindrome. Consistent with this observation, the point mutations in four base-pairs, GTTC, from +60 to +63 in the anterior half of the B block palindrome, has the most severe effect on transcription. In contrast, we showed that the central sequence and the posterior half (B2) played a stronger role than the anterior half (B1) of the B block palindrome in the interaction of the promoter with TFIIIC. This was corroborated by the observation that base substitutions in the central four base-pair sequence of the palindrome, TCGA, from +62 to +65, had the most severe effect on interaction with TFIIIC, and that mutations in most of the sequences in the posterior half of the B block palindrome had more drastic effects than mutations in the anterior half of the palindrome in this interaction. Furthermore, the spacing between the two halves of the B block palindrome had a drastic effect on the overall transcription efficiency and the interaction of the promoter with TFIIIC, suggesting that the interaction between the two halves of the B block palindrome is not only essential, but also synergistic for the interaction with TFIIIC as well as the assembly of a transcriptionally active preinitiation complex and efficient transcription.

Adenoviruses, Human↗
PubMed1985

Efficiency of in vitro transcription of Dictyostelium discoideum actin gene is affected by the nucleotide sequence of the transcription initiation region.

The actin gene of Dictyostelium discoideum is transcribed faithfully but with very low efficiency in a cell-free system containing HeLa cell extract [Takiya, S., Tabata, T., Iwabuchi, M., Hirose, S., & Suzuki, Y. (1984) J. Biochem. (Tokyo) 95, 1367-1377]. Using the same in vitro system, we determined that the promoter activity of the actin 5 gene is 100-200 times weaker than that of the silkworm fibroin gene. To clarify the cause of the low transcription efficiency, various chimeric genes were constructed from the actin and fibroin genes, and their transcription efficiencies were examined in vitro. Both the TATA box and the transcription initiation site of the two natural genes functioned in the transcription of the chimeric genes, the efficiency of which was especially affected by the transcription initiation region. In chimeric genes having the upstream sequence of the actin gene and a downstream sequence including the transcription initiation site of the fibroin gene, the transcription efficiency was higher than one-third of that of the natural fibroin gene. In chimeric genes having the actin transcription initiation region and an upstream sequence of the fibroin gene, the transcription efficiency was as low as that of the natural actin gene. We concluded that the transcription initiation site is a part of the promoter and an essential region for directing faithful and efficient initiation of gene transcription.

Actins↗
PubMed1990

Transcription of the testis-specific mouse protamine 2 gene in a homologous in vitro transcription system.

Transcriptionally active nuclear extracts were prepared from mouse testes to study the transcription of the testis-specific mouse protamine 2 (Prm-2) gene in vitro. The testicular system is unique among mammalian in vitro transcription systems in regard to its temperature optimum. In extracts made from prepuberal testes, the temperature optimum for in vitro transcription of Prm-2 is 30 degrees C, similar to somatic in vitro systems. However, in adult testis extracts, the optimum temperature for Prm-2 transcription is 20 degrees C. The different temperature optima seen in vitro for prepuberal and adult testes extracts parallels in vivo physiological temperature sensitivities of the differentiating male germ cells. The testis system also differs from other in vitro transcription systems in its divalent metal cation and ionic strength requirements for optimal transcription. The mouse Prm-2 gene is maximally transcribed at a MgCl2 concentration of 3-5 mM and over a KCl concentration range of 40-100 mM. By using the testis in vitro transcription system to study the Prm-2 gene by deletion analysis, we have determined that positive promotion for the gene lies within the region -170 to -82 from the start of transcription. This region contains a putative Sp-1 binding site. Additional upstream sequences appear to repress Prm-2 transcription in a heterologous transcription system.

Animals↗
PubMed1990

Effects of nutrients and hormones on transcriptional and post-transcriptional regulation of acetyl-CoA carboxylase in rat liver.

The effects of nutrients and hormones on transcriptional and post-transcriptional regulation of acetyl-CoA carboxylase in rat liver were investigated following a cDNA cloning. After refeeding a carbohydrate/protein diet to fasted rats, the transcriptional rate was increased 2.5-fold in only 1 h. The mRNA concentration reached a maximal level of 9-12-fold increase in 8-16 h, and the enzyme induction increased 10-fold in 48 h. By a carbohydrate diet without protein, the transcriptional rate, mRNA concentration and enzyme induction were similarly increased to the levels in the carbohydrate/protein diet. It appears that protein feeding is not necessary to induce acetyl-CoA carboxylase. Corn oil feeding decreased the transcriptional rate. In diabetic rats, the transcriptional rate, mRNA concentration and enzyme induction were very low in comparison with the normal. After insulin treatment, the transcriptional rate was increased 2-fold (the normal level) in 2 h in diabetic rats. By fructose feeding to diabetic rats, the transcriptional rate and mRNA concentration were increased similarly to the levels reached by insulin treatment, while the enzyme induction was increased by only 60%. Thus, it is suggested that insulin is importantly involved in the transcription and also translation of acetyl-CoA carboxylase. On the other hand, triiodothyronine treatment increased the mRNA and enzyme levels in diabetic and normal rats, and somewhat increased the transcriptional rate only in diabetic rats. Triiodothyronine appears to stabilize the mRNA besides having an insulin-like action in acetyl-CoA carboxylase transcription.

Acetyl-CoA Carboxylase↗
PubMed1990

Light-induced transcription of chloroplast genes. psbA transcription is differentially enhanced in illuminated barley.

Transcription and Northern dot blot analyses were used to determine the relationship between light-induced changes in RNA levels and transcription in barley and maize plastids. Overall plastid transcription activity is near maximum and largely light independent in apical sections of 4.5-day-old dark-grown barley seedings. With continued development in the dark, transcription activity in barley plastids declined precipitously, but transcription could be reactivated if plants were illuminated. Illumination of 8-day-old dark-grown barley seedlings increased the transcription of psbA, rbcL, and 16 S rDNA; however, the stimulation of psbA transcription was 2-4-fold more than rbcL or 16 S rDNA. Differential enhancement of psbA transcription occurred when 4.5- or 8-day-old dark-grown seedlings were illuminated, and the influence persisted in plants illuminated for 72 h. Illumination of 8-day-old dark-grown barley caused an increase in psbA and rbcL mRNA levels, but the increase in psbA mRNA was greater than that of rbcL. Similarly, illumination of 9-day-old dark-grown maize increased plastid transcription activity and the levels of psbA and rbcL mRNA. These results indicate that light can modulate overall plastid transcription activity and differentially stimulate the transcription of genes including psbA.

Blotting, Northern↗
PubMed1983

Release of transcript and template during transcription termination at the trp operon attenuator.

We studied release of trp leader RNA and trp template DNA from RNA polymerase during transcription termination at the attenuator of the trp operon of Escherichia coli. Preliminary evidence had suggested that a stable ternary complex was formed at the trp attentuator. We observed that the complexes between RNA polymerase and trp leader RNA and the DNA template produced during transcription were labile at high salt concentrations and were undetectable when transcription was performed in the presence of heparin. These characteristics are atypical of the stable transcription termination complexes described by others (Richardson, J. P., and Conaway, R. (1980) Biochemistry 19, 4293-4299; Shigesada, K., and Wu, C. (1980) Nucleic Acids Res. 8, 3355-3369). We successfully reconstituted polymerase-trp leader RNA complexes in simple mixing experiments; these and other studies indicated that it is core polymerase that binds the leader transcript and the DNA template. In agreement with this conclusion, it was observed that sigma factor inhibited binding of RNA polymerase to the trp leader transcript and the DNA template and displaced leader RNA from RNA polymerase during transcription. It seems likely that small amounts of core polymerase present in the holoenzyme preparation, or generated during transcription, are responsible for the nonspecific binding of RNA transcript and DNA template. Our findings, therefore, suggest that the transcription termination event at the trp attenuator normally involves spontaneous dissociation of polymerase, template, and RNA transcript.

DNA, Bacterial↗
PubMed1989

Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes.

Transcription complexes that assemble on tRNA genes in a crude Saccharomyces cerevisiae cell extract extend over the entire transcription unit and approximately 40 base pairs of contiguous 5'-flanking DNA. We show here that the interaction with 5'-flanking DNA is due to a protein that copurifies with transcription factor TFIIIB through several steps of purification and shares characteristic properties that are normally ascribed to TFIIIB: dependence on prior binding of TFIIIC and great stability once the TFIIIC-TFIIIB-DNA complex is formed. SUP4 gene (tRNATyr) DNA that was cut within the 5'-flanking sequence (either 31 or 28 base pairs upstream of the transcriptional start site) was no longer able to stably incorporate TFIIIB into a transcription complex. The TFIIIB-dependent 5'-flanking DNA protein interaction was predominantly not sequence specific. The extension of the transcription complex into this DNA segment does suggest two possible explanations for highly diverse effects of flanking-sequence substitutions on tRNA gene transcription: either (i) proteins that are capable of binding to these upstream DNA segments are also potentially capable of stimulating or interfering with the incorporation of TFIIIB into transcription complexes or (ii) 5'-flanking sequence influences the rate of assembly of TFIIIB into stable transcription complexes.

Base Sequence↗
PubMed1990

Interferon-mediated transcriptional and post-transcriptional modulation of complement gene expression in human monocytes.

The addition of lymphoblastoid interferon alpha, fibroblast interferon beta and recombinant interferon gamma to in vitro monocyte cultures produced dose-dependent increases in transcription rates of the genes encoding the second component of complement (C2), factor B (B) and C1 inhibitor, and the abundance of their respective mRNA. Interferon gamma was the most effective at stimulating transcription of the C1-inhibitor gene whereas interferons alpha and beta were more effective at increasing the transcription of the C2 and B genes. Transcription of the C3 gene was reduced by interferon gamma. None of these cytokines altered the level of transcription of the actin gene. Interferon-induced changes in the levels of transcription of the C2, B and C1-inhibitor genes occurred rapidly, with significant changes occurring within 30 min of exposure to these cytokines. Within 4 h of removal of the interferons from the culture fluid, the level of transcription of the C1-inhibitor, C2, B and C3 genes returned to control values, as did abundance of C2, B and C3 mRNA. However, the abundance of C1-inhibitor mRNA remained elevated in interferon-gamma-treated monocytes. Combinations of interferons produced less than additive effects on the stimulation of the transcription of C2, B and C1-inhibitor genes, whereas measurements of C1-inhibitor mRNA and B mRNA showed that interferon gamma acted synergistically with interferon gamma to increase the abundance of the mRNA. Their effects on C2 mRNA abundance were less than additive. The half-lives of C1-inhibitor, C2, B and C3 mRNA were not altered by interferon alpha, whereas interferon gamma shortened the half-life of C2 mRNA by approximately 50%, and prolonged the half-lives of B and C1-inhibitor mRNA approximately twofold and fivefold, respectively. The half-life of C3 mRNA was unaltered by either interferon. These results show that the large increase in C1-inhibitor synthesis which occurs in interferon-gamma-treated monocytes, is due to a combination of increased transcription and increased C1-inhibitor mRNA stability. They also suggest that the synergistic effects of interferon alpha together with interferon gamma on C1-inhibitor and factor B synthesis is also dependent upon increased transcription and increased mRNA stability.

Complement C1 Inactivator Proteins↗
PubMed1987

Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: inactivation of specific transcription factors.

The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoter, however, was not altered by infection of cells with the virus. We conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.

Cell Line↗
PubMed1991

The relative importance of transcriptional and post transcriptional regulation of Drosophila chorion gene expression during oogenesis.

To determine the relative roles of transcriptional and post-transcriptional events in establishing the temporal pattern of chorion gene expression in Drosophila, we have examined chorion gene transcription, RNA accumulation, and protein synthesis in follicles of selected pre-, early-, and late-choriogenic stages. Chorion gene transcription was assayed in follicle cell nuclei by nuclear run-on reactions. For the s15, s16, s18, s36, and s38 chorion genes, the periods of intense transcription are as predicted from the dynamics of RNA accumulation and protein synthesis, indicating that these genes are primarily regulated at the transcriptional level. In contrast, gene s19 appears subject to post-transcriptional control at stage 14, when transcription rates are substantially higher than predicted from the observed RNA levels. Transcription of regions between the clustered and tandemly oriented chorion genes was also examined. In contrast to many RNA polymerase II transcribed genes, for the s18 and s36 chorion genes run-on transcription appears to terminate within about 100 base pairs downstream of the polyadenylation sites, corroborating previous reports based on electron microscopy of s36 [Osheim et al., EMBO J 5:3591-3596, 1986].

Animals↗
PubMed1992

Dynamical behavior of psb gene transcripts in greening wheat seedlings. I. Time course of accumulation of the pshA through psbN gene transcripts during light-induced greening.

The time course of the accumulation of the transcripts from 13 psb genes encoding a major part of the proteins composing photosystem II during light-induced greening of dark-grown wheat seedlings was examined focusing on early stages of plastid development (0.5 h through 72 h). The 13 genes can be divided into three groups. (1) The psbA gene is transcribed as a single transcript of 1.3 kb in the dark-grown seedlings, but its level increases 5- to 7-fold in response to light due to selective increase in RNA stability as well as in transcription activity. (2) The psbE-F-L-J operon, psbM and psbN genes are transcribed as a single transcript of 1.1 kb, two transcripts of 0.5 and 0.7 kb and a single transcript of 0.3 kb, respectively, in the dark-grown seedlings. The levels of accumulation of every transcript remain unchanged or rather decrease during plastid development under illumination. (3) The psbK-I-D-C gene cluster and psbB-H operon exhibit fairly complicated northern hybridization patterns during the greening process. When a psbC or psbD gene probe was used for northern hybridization, five transcripts differing in length were detected in the etioplasts from 5-day old dark-grown seedlings. After 2 h illumination, two new transcripts of different length appeared. Light induction of new transcripts was also observed in the psbB-H operon.

Blotting, Northern↗
PubMed1992

In vivo transcriptional analysis of three baculovirus genes: evidence of homology between viral and host transcripts.

Transcripts for the gp64 and polyhedrin genes as well as a 1629 open reading frame (1629 ORF) of the Autographa californica nuclear polyhedrosis virus were examined in the midgut tissues and hemocytes of uninfected and infected host Trichoplusia ni larvae and Sf21 cells. Polyhedrin-specific transcripts of 1.2, 3.4, and 4.9 kb were expressed in both infected larval tissues and infected Sf21 cells. The highest level of expression for polyhedrin-specific transcripts was observed in hemocytes, whereas the lowest level occurred in midgut. The expression of the 2.0-kb transcript for the gp64 gene increased continuously through 72 hr postinfection in the infected midgut tissues. This transcript was also observed in infected hemocytes, though its expression declined at 72 hr postinfection, as the expression for polyhedrin-specific transcripts peaked. The 1629 ORF transcripts of 2.0 and 3.2 kb were expressed in both types of infected tissues. More significantly, a 1629-ORF-specific probe detected host transcripts of 0.7 and 2.5 kb in uninfected midgut tissues as well as transcripts of 2.5, 8.8, and 11.0 kb in uninfected hemocytes under high stringency conditions. The latter results indicate that these host transcripts share homology with the 1629 ORF gene.

Animals↗
PubMed1979

Expression of the autonomous parvovirus H1 genome: evidence for a single transcriptional unit and multiple spliced polyadenylated transcripts.

We identified viral transcripts in parvovirus H1-infected rodent cells using the S1 nuclease mapping technique of Berk and Sharp (1977, 1978). The most abundant viral transcript, present in both nucleus and cytoplasm, is approximately 2.8 kb long and represents about 56% of the viral genome. Less abundant viral transcripts of 3.0, 1.45 and 1.30 kb, and possibly other minor viral transcripts, are also detected in nuclear and cytoplasmic fractions. In contrast, a prominent 4.7 kb viral transcript which corresponds to 95% of the viral DNA is found only in the nucleus; this finding suggests that the parvovirus genome may function as a single transcription unit. Virus-infected cells pretreated with cycloheximide accumulate all these viral transcripts. Analyses of RNA-DNA hybrids (isolated from neutral agarose gels) by electrophoresis on alkaline agarose gels indicate that the 4.7, 3.0 and 2.8 kb viral transcripts are "spliced" RNAs. The nuclear-specific 4.7 kb transcript appears to be encoded by two noncontiguous DNA segments of 2.2 and 2.6 kb. The 3.0 and 2.8 kb transcripts are apparently encoded by.a 2.6 kb segment of DNA and one or more much smaller noncontiguous DNA segments, one of which is approximately 170 nucleotides long.

Animals↗
PubMed1991

Contributory effects of de novo transcription and premature transcript termination in the regulation of human epidermal growth factor receptor proto-oncogene RNA synthesis.

Overexpression of the epidermal growth factor (EGF) receptor (c-erbB) proto-oncogene is a frequent occurrence in human carcinoma and appears to accompany autocrine or paracrine transforming growth factor-alpha expression, which in model systems can result in activation of EGF receptor tyrosine kinase activity and phenotypic transformation. Here we have investigated the transcriptional regulation of the EGF receptor gene, by run-on transcription in isolated nuclei derived from epithelioid tumor lines. The level of transcription was measured at various points on the 100-kilobase pair EGF receptor gene locus, on either sense or antisense DNA strands. We find the level of sense strand transcription along exon 1 is 8-fold higher than transcription in exons 2-26. Primary EGF receptor transcripts appear to pause or terminate prematurely between exons 1 and 2. Termination was mapped to a sequenced region approximately 2 kilobase pairs 3' of exon 1, proximal to a previously reported DNase I hypersensitive site and an enhancer-like activity. Transcription in the CpG-rich region surrounding exon 1 is bidirectional, with antisense transcripts initiating in intron 1 and extending through the coding first exon. Activation of protein kinase C results in a 5-fold induction of EGF receptor transcription, accompanied by a slow release in the block RNA elongation between exon 2 and exon 26, showing that EGF receptor RNA synthesis may be altered by changes in de novo transcription and by a block to RNA elongation.

Amino Acid Sequence↗
PubMed1988

Transcriptional regulation of a plasminogen activator gene by cyclic AMP in a homologous cell-free system. Involvement of cyclic AMP-dependent protein kinase in transcriptional control.

We have developed a homologous cell-free transcription system using extracts from the porcine kidney cell line LLC-PK1 to study the molecular mechanisms by which cAMP regulates urokinase-type plasminogen activator (uPA) gene transcription. We demonstrated accurate initiation of transcription using a cloned fragment of the uPA gene as template. The in vitro transcription rate was stimulated by up to 10-fold by the addition of cAMP (greater than 10 microM). This effect of cAMP on the transcription was greater for closed circular than for linear templates. Furthermore, addition of the purified catalytic subunit of cAMP-dependent protein kinase stimulated the in vitro transcription in the absence of cAMP to levels 2-fold higher than those observed with cAMP. Addition of cAMP had no stimulatory effect on the transcription of the rat heme oxygenase gene promoter tested under identical conditions. HeLa whole cell extract by itself showed no stimulation of transcription of the uPA gene by cAMP. Results of reconstitution experiments using HeLa whole cell extracts and nuclear lysates from LLC-PK1 cells suggest the presence of putative cAMP regulatory factor(s) as well as general transcription factor(s) in the nucleus of LLC-PK1 cells. These results provide experimental evidence directly implicating cAMP-dependent protein kinase in the regulation of gene transcription.

Animals↗
PubMed1992

Yeast transcription factor IID participates in cell-free transcription of a mammalian ribosomal protein TATA-less promoter.

We analysed transcription of the gene for the ribosomal protein (rp) L32 of the mouse, which is transcribed in mouse L1210 nuclear extracts in vitro. The rpL32 gene lacks a canonical TATA box. Hence it has been suggested that this gene has an alternative transcription pathway not requiring transcription factor IID (TFIID). Selective inactivation of TFIID in nuclear extract completely abolished the transcription of rpL32 in vitro. Selective inactivation was restored by the addition of cloned and purified yeast TFIID (yTFIID), indicating that this TATA-less rpL32 promoter utilizes TFIID for its transcription initiation. Furthermore, addition of an oligonucleotide-containing TATA sequence interfered with the rpL32 transcription and this was overcome by the addition of yTFIID. To further examine the stage of involvement of TFIID in rpL32 transcription, TATA oligonucleotide was added to nuclear extract before and after the formation of the transcription complex. The results reveal that TFIID associates with the pre-initiation complex and that this complex is largely resistant to added TATA oligonucleotide. Our results show, for the first time, that the TATA-less rpL32 gene utilizes TFIID for transcription initiation.

Animals↗