Gene machines

Abstract:

In order to define the mean DNA bend angle and distribution of DNA bend angles in the catabolite activator protein (CAP)-DNA complex in solution under standard transcription initiation conditions, we have performed nanosecond time-resolved fluorescence measurements quantifying energy transfer between a probe incorporated at a specific site in CAP, and a complementary probe incorporated at each of five specific sites in DNA. The results indicate that the mean DNA bend angle is 77(+/-3) degrees - consistent with the mean DNA bend angle observed in crystallographic structures (80(+/-12) degrees ). Lifetime-distribution analysis indicates that the distribution of DNA bend angles is relatively narrow, with <10 % of DNA bend angles exceeding 100 degrees. Millisecond time-resolved luminescence measurements using lanthanide-chelate probes provide independent evidence that the upper limit of the distribution of DNA bend angles is approximately 100 degrees. The methods used here will permit mutational analysis of CAP-induced DNA bending and the role of CAP-induced DNA bending in transcriptional activation.

Abstract:

Using fluorescence resonance energy transfer, we show that, in the majority of transcription complexes, sigma(70) is not released from RNA polymerase upon transition from initiation to elongation, but, instead, remains associated with RNA polymerase and translocates with RNA polymerase. The results argue against the presumption that there are necessary subunit-composition differences, and corresponding necessary mechanistic differences, in initiation and elongation. The methods of this report should be generalizable to monitor movement of any molecule relative to any nucleic acid.

Abstract:

A sequence element located immediately upstream of the TATA element, and having the consensus sequence 5'-G/C-G/C-G/A-C-G-C-C-3', affects the ability of transcription factor IIB to enter transcription complexes and support transcription initiation. The sequence element is recognized directly by the transcription factor IIB. Recognition involves alpha-helices 4' and 5' of IIB, which comprise a helix-turn-helix DNA-binding motif. These observations establish that transcription initiation involves a fourth core promoter element, the IIB recognition element (BRE), in addition to the TATA element, the initiator element, and the downstream promoter element, and involves a second sequence-specific general transcription factor, IIB, in addition to transcription factor IID.

Abstract:

Adenovirus large E1A, Epstein-Barr virus Zebra, and herpes simplex virus VP16 were studied as models of animal cell transcriptional activators. Large E1A can activate transcription from a TATA box, a result that leads us to suggest that it interacts with a general transcription factor. Initial studies showed that large E1A binds directly to the TBP subunit of TFIID. However, analysis of multiple E1A and TBP mutants failed to support the significance of this in vitro interaction for the mechanism of activation. Recent studies to be reported elsewhere indicate that conserved region 3 of large E1A, which is required for its activation function, binds to one subunit of a multisubunit protein that stimulates in vitro transcription in response to large E1A and other activators. A method was developed for the rapid purification of TFIID approximately 25,000-fold to near homogeneity from a cell line engineered to express an epitope-tagged form of TBP. Purified TFIID contains 11 major TAFs ranging in mass from approximately 250 to 20 kD. Zta and VP16, but not large E1A, greatly stimulate the rate and extent of assembly of a TFIID-TFIIA complex on promoter DNA (DA complex). For VP16, this is a function of the carboxy-terminal activation subdomain. An excellent correlation was found between the ability of VP16C mutants to stimulate DA complex assembly and their ability to activate transcription in vivo. Consequently, for a subset of activation domains, DA complex assembly activity is an important component of the overall mechanism of activation.