Biomedical subjects

K U Sprague

Publications and source records attributed to K U Sprague.

At least 19 recordsLinked to original sources

PubMed2000

TATA-Binding protein-TATA interaction is a key determinant of differential transcription of silkworm constitutive and silk gland-specific tRNA(Ala) genes.

We have investigated the contribution of specific TATA-binding protein (TBP)-TATA interactions to the promoter activity of a constitutively expressed silkworm tRNA(C)(Ala) gene and have also asked whether the lack of similar interactions accounts for the low promoter activity of a silk gland-specific tRNA(SG)(Ala) gene. We compared TBP binding, TFIIIB-promoter complex stability (measured by heparin resistance), and in vitro transcriptional activity in a series of mutant tRNA(C)(Ala) promoters and found that specific TBP-TATA contacts are important for TFIIIB-promoter interaction and for transcriptional activity. Although the wild-type tRNA(C)(Ala) promoter contains two functional TBP binding sequences that overlap, the tRNA(SG)(Ala) promoter lacks any TBP binding site in the corresponding region. This feature appears to account for the inefficiency of the tRNA(SG)(Ala) promoter since provision of either of the wild-type TATA sequences derived from the tRNA(C)(Ala) promoter confers robust transcriptional activity. Transcriptional impairment of the wild-type tRNA(SG)(Ala) gene is not due to reduced incorporation of TBP into transcription complexes since both the tRNA(C)(Ala) and tRNA(SG)(Ala) promoters form transcription complexes that contain the same amount of TBP. Thus, the deleterious consequences of the lack of appropriate TBP-TATA contacts in the tRNA(SG)(Ala) promoter must come from failure to incorporate some other essential transcription factor(s) or to stabilize the complete complex in an active conformation.

Animals↗

PubMed1999

A TATA element is required for tRNA promoter activity and confers TATA-binding protein responsiveness in Drosophila Schneider-2 cells.

In contrast to yeast and mammalian systems, which depend principally on internal promoter elements for tRNA gene transcription, insect systems require additional upstream sequences. To understand the function of the upstream sequences, we have asked whether the Bombyx mori tRNACAla and tRNASGAla genes, which are absolutely dependent on these sequences in vitro, also require them for transcription in vivo. We introduced wild-type and mutant versions of the Bombyx tRNAAla genes into Drosophila Schneider-2 cells and found that the tRNACAla gene is efficiently transcribed and that its transcription depends strongly on the distal segment of its upstream promoter. In contrast, the tRNASGAla gene is inefficiently transcribed, and this inefficiency results from lack of a specific sequence within the distal tRNACAla upstream promoter. This sequence, 5'-TTTATAT-3', is sufficient to increase the activity of the tRNASGAla promoter to that of the tRNACAla promoter. Moreover, promoters containing the 5'-TTTATAT-3' element are stimulated by increased levels of cellular TATA-binding protein. Together these results indicate that, in insect cells, a TATA-like element is specifically required to form functional TATA-binding protein-containing complexes that promote efficient transcription of tRNA genes.

Animals↗

PubMed1998

Cloning and characterization of the TATA-binding protein of the silkworm Bombyx mori.

The TATA-binding protein is a general transcription factor required by all three eukaryotic nuclear RNA polymerases. In order to study the function of this protein in the transcription of tRNA genes in the silkworm Bombyx mori, we have cloned TBP cDNA from a silkworm cDNA library. As in most other eukaryotes, TBP in silkworms is encoded by a single copy gene and contains a highly conserved C-terminal domain that includes a basic region and two direct repeats. In the less conserved N-terminal domain, silkworm TBP exhibits characteristics such as a glutamine-rich stretch and three imperfect Pro-Met-Thr-like repeats that are also found in Drosophila and human TBP. Silkworm TBP expressed in Escherichia coli and purified to apparent homogeneity binds the TATA element of the wild-type adenovirus major late promoter with nanomolar affinity.

Amino Acid Sequence↗

PubMed1996

Silkworm TFIIIB binds both constitutive and silk gland-specific tRNA Ala promoters but protects only the constitutive promoter from DNase I cleavage.

We have identified a complex between TFIIIB and the upstream promoter of silkworm tRNA Ala genes that is detectable by gel retardation and DNase I footprinting. Formation of this complex depends on the integrity of previously identified upstream promoter elements and on the presence of other silkworm transcription factors, either TFIIID or a fraction that contains both TFIIIC and TFIIID. We have used this complex to compare the interactions of TFIIIB with two kinds of tRNA Ala genes whose different in vitro transcription properties are conferred by the upstream segments of their promoters. These are the tRNA C Ala genes, which are transcribed constitutively, and the tRNA SG Ala genes, which are transcribed only in the silk gland. We find that TFIIIB binds tRNA SG Ala genes with lower affinity than it binds tRNA C Ala genes. In addition, the TFIIIB complex formed on tRNA SG Ala genes differ qualitatively from those formed on tRNA C Ala genes. Both the transcriptional activity of tRNA SG Ala complexes and the ability of the complexes to protect upstream DNA from DNase I digestion are reduced.

Animals↗

PubMed1995

Silkworm TFIIIA requires additional class III factors for commitment to transcription complex assembly on a 5S RNA gene.

We find striking similarities in promoter structure and requirements for template commitment on 5S RNA and tRNA genes from silkworms. The promoters are nearly the same size (approximately 160 bp) and include flanking as well as internal sequences. To analyze the factor requirements for 5S RNA transcription complex assembly in a completely homologous system, we have isolated a silkworm fraction that is highly enriched for the 5S RNA-specific transcription factor, TFIIIA. Using this fraction, together with the other silkworm fractions, TFIIIB, TFIIIC, TFIIID and RNA polymerase III, we demonstrate that the requirements for 5S RNA transcription complex assembly are very similar to those previously established for a tRNA(C)(Ala) gene. Specifically, no individual factor fraction is sufficient for commitment of silkworm 5S RNA genes to transcription complex assembly. Rather, combinations of at least three factor fractions are required. Our observation that more than one subset of factors is competent for commitment suggests that silkworm 5S RNA genes further resemble tRNA(C)(Ala) genes in their ability to use multiple pathways for transcription complex formation.

Animals↗

PubMed1994

Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions.

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

Animals↗

PubMed1994

TFIIIR is an isoleucine tRNA.

Promoter-specific transcription by silkworm RNA polymerase III is dependent on several transcription factors (TFs) in addition to the polymerase itself. The activities present in silk gland nuclear extracts that are necessary to reconstitute transcription from class III genes in vitro have been resolved into several partially purified components. These include TFIIIR, which is unusual because it is composed of RNA. Here, we identify the RNA that provides TFIIIR activity as silkworm tRNA(IleIAU). This conclusion is based on copurification of tRNA(IleIAU) with TFIIIR activity, TFIIIR activity in synthetic tRNA(Ile), and hybrid selection of TFIIIR activity by antisense tRNA(IleIAU). We have tested the ability of a variety of other tRNAs to stimulate transcription and find that TFIIIR activity is highly specific to silkworm tRNA(IleIAU).

Animals↗

PubMed1994

tRNA(IleIAU) (TFIIIR) plays an indirect role in silkworm class III transcription in vitro and inhibits low-frequency DNA cleavage.

tRNA(IleIAU) provides an activity, originally called TFIIIR, necessary to reconstitute transcription by silkworm RNA polymerase III in vitro from partially purified components. Here we report studies on the role of tRNA(IleIAU) in in vitro transcription. We show that tRNA(IleIAU) does not act positively but, rather, is required to prevent the action of a transcriptional inhibitor. We also show that the presence of tRNA(IleIAU) in transcription reaction mixtures prevents low-frequency DNA cleavage by the TFIIIB fraction. Studies on the mechanism of transcriptional inhibition suggest that this DNA cleavage could cause transcriptional inhibition through trans-inactivation of transcription machinery. The ability to block DNA cleavage, like the ability to facilitate transcription, is highly specific to silkworm tRNA(IleIAU).

Animals↗

PubMed1993

Transcription of a silkworm tRNA(cAla) gene is directed by two AT-rich upstream sequence elements.

A region within 35 nucleotides upstream of the transcription initiation site of a variety of silkworm Class III templates is absolutely required for transcription in vitro. To determine whether the activity of this region can be attributed to a particular sequence element, we systematically replaced 4-5 bp segments of the region upstream of a silkworm tRNA(cAla) gene. We show that replacement of either of two AT-rich blocks markedly impairs promoter function, whereas replacement of other sequences has little or no effect. Additional mutants were constructed to test whether base composition or sequence is important for function of the AT blocks. We find that some sequences are more effective than others, but that various AT-rich sequences can direct transcription at a high level. Possible mechanisms by which such elements could act are discussed.

Animals↗

PubMed1993

A novel RNA polymerase III transcription factor fraction that is not required for template commitment.

We have identified and partially characterized a novel class III transcription factor fraction (TFIIIE) from yeast nuclear extracts. TFIIIE is functionally distinct from the standard yeast transcription factor fractions, TFIIIB and TFIIIC. It is also different from either of the TFIIIB subfractions, B' and B". TFIIIE is essential for specific transcription of both tRNA and 5 S RNA genes, its activity is sensitive to proteinase K, and it exhibits an apparent sedimentation coefficient of 4.0 S when analyzed on glycerol gradients. In the case of a tRNA gene, TFIIIE does not play a role in the formation of stable preinitiation complexes containing TFIIIB and TFIIIC. It is required for single as well as multiple rounds of transcription, however. Thus, TFIIIE is involved in the utilization of stable transcription complexes, but its action is not restricted to reinitiation events.

Cell Nucleus↗

PubMed1992

New twists in class III transcription.

Recent work emphasizes the similarity between polymerase II and III in the mechanisms of transcription. Highlights of the past year include the alignment of individual polypeptides within class III transcription complexes and the demonstration that class III transcription machinery includes TFIID and an RNA component.

Animals↗

PubMed1991

A class III transcription factor composed of RNA.

It is generally assumed that the machinery that transcribes genes is composed entirely of polypeptides. However, in vitro transcription by silkworm RNA polymerase III requires a transcription factor that is not a polypeptide. This component, TFIIIR, is distinct from the previously identified transcription components: RNA polymerase III, and the accessory factors TFIIIA, TFIIIB, TFIIIC, and TFIIID. The newly discovered TFIIIR is a macromolecule that appears to be composed of RNA. It is resistant to heat, detergent, phenol, protease, and deoxyribonuclease, but it is sensitive to alkali and ribonuclease.

Animals↗

PubMed1991

Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors.

We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNA(Ala)C gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNA(Ala)C promoter is in contact with factors. The protected region extends from -1 to at least +136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequence-specific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNA(Ala)C promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes.

Animals↗

PubMed1988

Silk gland-specific tRNA(Ala) genes are tightly clustered in the silkworm genome.

To understand the basis for tissue-specific production and accumulation of alanine tRNA in silkworms, we have examined the organization of the genes that code for silk gland-specific and constitutive alanine tRNAs. We have found that all of the silk gland-specific tRNA(Ala) genes (approximately 20) appear to be tightly clustered at a single locus in the Bombyx genome. These genes are arranged in tandem at intervals of approximately 150 base pairs. In contrast to the arrangement of the silk gland-specific tRNA(Ala) genes, most of the 20 to 30 constitutive tRNA(Ala) genes are dispersed in the genome. Silk gland-specific tRNA(Ala) genes are not amplified or grossly rearranged in the silk gland. Thus it is likely that differential transcription, rather than changes in gene number or structure, accounts for the tissue-specific accumulation of tRNA(Ala).

Animals↗

PubMed1988

Transcriptional properties of BmX, a moderately repetitive silkworm gene that is an RNA polymerase III template.

We analyzed the transcriptional properties of a repetitive sequence element, BmX, that belongs to a large gene family (approximately 2 x 10(4) copies) in the genome of the Bombyx mori silkworm. We discovered BmX elements because of their ability to direct transcription by polymerase III in vitro and used them to test the generality of the properties of previously identified silkworm polymerase III control elements. We found that the signals that act in cis to control BmX transcription strongly resemble those that direct transcription of other silkworm polymerase III templates. As with silkworm tRNA and 5S RNA genes, transcription of BmX requires sequence signals located both upstream and downstream from the site of transcription initiation. The critical upstream sequences are structurally as well as functionally similar in the three kinds of templates. The downstream control region of BmX resembles the corresponding part of a silkworm alanine tRNA gene in that it provides a large (greater than 100 base pairs) region that influences transcription factor binding. Moreover, the factor-binding regions of both tRNA(Ala) and BmX genes are remarkable in that under certain conditions, key elements within them (the B boxes, for example) appear dispensable. This behavior can be understood if, in both of these templates, the downstream control region acts as a large target for interaction with a multifactor complex.

Animals↗

PubMed1987

The properties of a new polymerase III transcription factor reveal that transcription complexes can assemble by more than one pathway.

We have resolved a previously unidentified factor (TFIIID) that is required for in vitro transcription of polymerase III templates. Our ability to resolve factor D from each of the other components of the transcription machinery (polymerase and transcription factors IIIB and IIIC) allowed us to test the capacity of these separated components to form stable complexes with tRNA genes. We find that none of the individual components binds detectably to tRNA genes, but that certain combinations of transcription factors do bind. Our results show that TFIIID is essential for binding and that formation of a full transcription complex can proceed by either of two different pathways.

Animals↗

PubMed1986

Upstream sequences confer distinctive transcriptional properties on genes encoding silkgland-specific tRNAAla.

To understand the molecular basis for the tissue-specific accumulation of alanine tRNAs in silkworms, we have compared the transcriptional properties of genes that encode constitutive and silkgland-specific type alanine tRNA. Genes for each class of alanine tRNA behave very differently when used as templates for homologous in vitro transcription. Since these properties are likely related to the activities of the corresponding genes in vivo, we wished to identify the cis-acting elements responsible for them. We have therefore constructed hybrid silkgland-specific/constitutive genes and have analyzed their capacity to direct transcription in vitro. We find a simple pattern: the distinctive transcriptional properties of the two kinds of tRNAAla genes are the result of different positive signals upstream from their sites of transcription initiation.

Alanine↗

PubMed1985

A large region controls tRNA gene transcription.

We find that tRNA gene transcription is controlled by a region much larger than the previously described internal segments known as A and B boxes. In a Bombyx mori silkworm tRNA2Ala gene, transcriptionally important sequences extend at least from -13 to +146, and thus include sequences both upstream and downstream from the 98 base-pair primary transcription unit. We show that the apparent size of this control region can be manipulated by the conditions used to measure transcription in vitro. Specifically, the use of high concentrations of templates to overcome the effect of an inhibitory substance in crude extracts can make the control region appear smaller. We propose that this finding explains much of the observed variability in the sequence requirements for transcription of different tRNA genes. If so, large size is likely to be a general feature of tRNA gene control regions.

Animals↗