The two Saccharomyces cerevisiae SUA7 (TFIIB) transcripts differ at the 3'-end and respond differently to stress.

Despite much information as to the structure and function of the general transcription factors, little is known about the regulation of their expression. Transcription of the Saccharomyces cerevisiae SUA7 (TFIIB) gene results in the formation of two discrete transcripts. It was originally reported that the two transcripts were derived from two promoters separated by approximately 80 bp. We have found that the two transcripts are instead derived from a common promoter and differ at the 3'-end by approximately 115 bp. The longer of the two transcripts has an unusually long 3'-untranslated region. We have analyzed the levels of these transcripts under different cell growth conditions and find that the relative amounts of the two transcripts vary. Approximately equal amounts of each transcript are observed during exponential growth, but stresses and growth limiting conditions lead to a decrease in the relative amount of the larger transcript. These results suggest that the expression of the SUA7 gene may be controlled by regulation of 3'-end formation or mRNA stability. One of the general transcription factors, then, may be subject to regulation by a general response of the mRNA processing machinery.

Contributions of the TATA box sequence to rate-limiting steps in transcription initiation by RNA polymerase II.

We have examined the role of the TATA box in determining transcription initiation frequency in vitro by studying a collection of promoters containing different TATA sequences in the context of the adenovirus major late promoter. In addition to measuring transcription rates, we have determined how the sequence changes affected the association and dissociation kinetics and the affinity of TBP binding. We observed that transcription from promoters containing the highest affinity TATA boxes is limited by the rate with which TBP associates with the promoter. In contrast, transcription from promoters containing lower affinity TATA boxes appears to be limited both by how much TBP is bound and by the relatively low occupancy of the conformation that can undergo subsequent steps in preinitiation complex assembly. The implications of these results in understanding the mechanism of transcription enhancement by transcriptional activators is discussed.

DNA bending is an important component of site-specific recognition by the TATA binding protein.

We have used gel electrophoretic methods to analyze the extent, location and direction of the DNA bend induced by the TATA binding protein (TBP) upon binding to a consensus TATA box sequence. Our observations were consistent with the proposed models for the X-ray crystal structure of the TBP-TATA box complex. We have also measured the magnitude and direction of the bend induced by TBP upon binding a number of variant TATA box sequences for which we have measured TBP binding affinity. We found that the extent to which the DNA was bent in the complex differed among the various sequences and was correlated with the stability of the complex; that is, the greater the stability of the complex, the more the DNA appeared to be bent by TBP. This study provides the first evidence that the structure of the TBP-DNA complex may vary with different DNA sequences. In addition, we propose, based on our findings, that the energetics of bending contribute significantly to the overall binding affinity of TBP for different sequences.

Kinetic analysis of yeast TFIID-TATA box complex formation suggests a multi-step pathway.

The eukaryotic transcription factor TFIID recognizes and binds a promoter sequence element called the TATA box. We have analyzed the interaction of yeast TFIID with the consensus TATA box sequence of the adenovirus major late promoter. To facilitate this detailed characterization, we developed a method for obtaining quantitative information from a gel retardation (bandshift) assay, allowing measurement of the rate and extent of TFIID-TATA box complex formation. Using this assay and DNase I protection assays, we determined that the association rate constant for TFIID binding to the major late promoter was too low to be consistent with a simple diffusion-limited association, suggesting that the binding proceeds by a multi-step pathway. Furthermore, we found that the slow rate of TFIID binding reported by other research groups was not the consequence of a rate-limiting conformational change, as has been previously suggested. Instead, we observed that the formation of a stable TFIID-TATA box complex was relatively rapid (complete in less than 1 min) at saturating concentrations of TFIID. We have proposed a two-step pathway consistent with the observed kinetics and have considered the possible contributions of each step to the overall rate of TFIID binding. This study lays the groundwork for a systematic characterization of the interaction of TFIID with additional TATA box sequences, including an experimental test of the possibility that different steps in the binding reaction are rate-limiting for different promoters.

IS10 transposition is regulated by DNA adenine methylation.

We show that dam- mutants are a major class of E. coli mutants with increased IS10 activity. IS10 has two dam methylation sites, one within the transposase promoter and one within the inner terminus where transposase presumably binds. Absence of methylation results in increased activity of both promoter and terminus, and completely accounts for increased transposition in dam- strains. Transposition of Tn903 and Tn5 are also increased in dam- strains, probably for analogous reasons. Transposition is also increased when IS10 is hemimethylated. One hemimethylated species is much more active than the other and is estimated to be at least 1000 times more active than a fully methylated element. Evidence is presented that the promoter and inner terminus of IS10 are coordinately activated in a dam-dependent fashion, presumably because they are hemimethylated at the same time. Thus, in dam+ strains, IS10 will transpose preferentially when DNA is hemimethylated. We suggest specifically that IS10 transposition may preferentially occur immediately after passage of a chromosomal replication fork.

A cII-dependent promoter is located within the Q gene of bacteriophage lambda.

We have found a cII-dependent promoter, PaQ, within the Q gene of bacteriophage lambda. Transcription experiments and abortive initiation assays performed in vitro showed that the promoter strength and the cII affinity of PaQ were comparable to the other cII-dependent lambda promoters, PE and PI. The location and leftward direction of PaQ suggests a possible role in the delay of lambda late-gene expression by cII protein, a phenomenon that has been called cII-dependent inhibition. We have constructed a promoter down mutation, paq-1, by changing a single base pair in the putative cII binding site of the promoter by oligonucleotide site-directed mutagenesis. The paq-1 mutant promoter required about 4-fold higher cII concentrations for maximal activation compared to the wild-type PaQ. We tested the hypothesis that PaQ is responsible in part for the delay of lambda late-gene expression by recombining the paq-1 mutation into a phage showing severe cII-dependent inhibition. We found that the paq-1 mutation relieved the cII-dependent growth defect of this phage. The paq-1 mutation (in combination with lambda cI857) resulted in a clear-plaque phenotype at the permissive temperature of 32 degrees C. The role of the PaQ-initiated antisense transcript in the control of lambda development is discussed.

Three promoters near the termini of IS10: pIN, pOUT, and pIII.

We have identified three IS10-encoded promoters, pIN, the promoter for IS10's transposase gene, is intrinsically weak, contributing to the low frequency of IS10 transposition in vivo. Its transcripts begin near the "outside" end of IS10 and extend inward across the element. pOUT, a strong promoter just internal to and opposing pIN, directs transcription outward. Its transcripts are proposed to inhibit translation of the transposase gene in trans (accompanying paper). pOUT may also inhibit transcription from pIN in cis. pIII, a weak promoter near the "inside" end of IS10, is of unknown genetic importance. Many transposable elements activate, by adjacent insertion, silent genes lacking normal promoters. Such IS10-promoted turn-on is mediated by pOUT and results from continuation of pOUT-initiated transcripts past the IS10 terminus, into adjoining chromosomal material. Wild-type and mutant IS10 promoters have been analyzed in vitro. pIN is weaker than pOUT because of inefficient isomerization from closed to open complexes. Despite their proximity, pIN and pOUT do not interact before or during open complex formation.

Studies on the selectivity of DNA precipitation by spermine.

We have examined the selectivity of the precipitation of DNA by spermine. We have found that the intra- and intermolecular condensation of DNA induced by spermine is highly selective even in the presence of added protein or triphosphates. We have also investigated the influence of buffer components on the threshold concentration of spermine required for DNA precipitation. Representative applications exploiting the selectivity of the precipitation reaction are also described.