High-Mobility-Group Proteins NHP6A and NHP6B Participate in Activation of the RNA Polymerase IIISNR6 Gene

Abstract

Transcription of yeast class III genes involves the formation of a transcription initiation complex that comprises RNA polymerase III (Pol III) and the general transcription factors TFIIIB and TFIIIC. Using a genetic screen for positive regulators able to compensate for a deficiency in a promoter element of the SNR6 gene, we isolated the NHP6A and NHP6B genes. Here we show that the high-mobility-group proteins NHP6A and NHP6B are required for the efficient transcription of the SNR6 gene both in vivo and in vitro. The transcripts of wild-type and promoter-defectiveSNR6 genes decreased or became undetectable in annhp6AΔ nhp6BΔ double-mutant strain, and the protection over the TATA box of the wild-type SNR6 gene was lost in nhp6AΔ nhp6BΔ cells at 37°C. In vitro, NHP6B specifically stimulated the transcription of SNR6 templates up to fivefold in transcription assays using either cell nuclear extracts from nhp6AΔ nhp6BΔ cells or reconstituted transcription systems. Finally, NHP6B activated SNR6transcription in a TFIIIC-independent assay. These results indicate that besides the general transcription factors TFIIIB and TFIIIC, additional auxilliary factors are required for the optimal transcription of at least some specific Pol III genes.

ACKNOWLEDGMENTS

We are very grateful to Reid Johnson for his generous gift of the Y865 and Y869 yeast strains, of the RJ1963 and RJ1964 E. coli strains, and of the antibodies against NHP6A and NHP6B. We warmly thank Giorgio Dieci for providing us with the recombinant TBP, TFIIIB70, and TFIIIB90 purified proteins and with the KS-tDNAIle(TAT)199 and KS-tDNAIle(TAT)36 plasmids harboring the I(TAT)LR1 and I(TAT)DR2 genes; Emmanuel Favry for excellent technical assistance; and Olivier Lefebvre and Christine Conesa for stimulating discussions.

S.L. was supported by a Commissariat à l'Energie Atomique postdoctoral fellowship. F.T. and M.L. were supported by the Swiss National Science Foundation.