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Volume 271, Number 51, Issue of December 20, 1996 pp. 33039-33046
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Mapping the Contacts of Yeast TFIIIB and RNA Polymerase III at Various Distances from the Major Groove of DNA by DNA Photoaffinity Labeling

(Received for publication, May 29, 1996, and in revised form, October 7, 1996)

From the Department of Medical Biochemistry, School of Medicine, Southern Illinois University at Carbondale, Carbondale, Illinois 62901-4413

The structure of the Saccharomyces cerevisiae RNA polymerase III transcription complex on the SUP4 tRNA^Tyr gene was probed at distances of ~10 to ~23 Å from the C-5 methyl of thymidine in the major groove of DNA using photoreactive aryl azides attached to deoxyuridine by variable chain lengths. The nucleotide analogs contained an azidobenzoyl group attached with chain lengths that were incrementally increased by ~4.3 Å by inserting 1-3 glycine residues into the chain. Another photoreactive deoxyuridine analog was made that contained a butyl chain (ABU-dUMP) to assess the effect of the chain's hydrophobicity on its ability to photoaffinity label the transcription complex. These nucleotide analogs were incorporated at base pairs (bp) 26/21, 17, or 3/2 on the nontranscribed strand of the SUP4 tRNA^Tyr gene along with an [-³²P]dNMP by primer extension using an immobilized single-stranded DNA template annealed to specific oligonucleotides.

The 27-kDa subunit of TFIIIB or the TATA box binding protein was photoaffinity labeled at bp 26/21 with nucleotide analogs containing a ~19- or ~23-Å chain and not with shorter chains of ~10 to ~15 Å in length. The B" subunit of TFIIIB (M_r = 90 kDa) was photoaffinity labeled at bps 26/21 with DNA containing a ~14-Å chain and not with shorter or longer chains. Cross-linking of the B" subunit was inhibited by binding of RNA polymerase III (Pol III) to the TFIIIB-DNA complex and suggested that Pol III binding causes a conformational change in the TFIIIB-DNA complex resulting in the displacement of the 90-kDa subunit at bps 26/21. Next, the chain length dependence of photoaffinity labeling the 34-kDa subunit of Pol III at bps 17 and 3/2 indicated that the 34-kDa subunit of Pol III is slightly removed from the major groove at bp 17 in the initiation complex and makes closer contact at bps 3/2 in a stalled elongation complex.

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