Vol. 14, No. 6, pp. 731-739, March 15, 2000

RESEARCH PAPER
The carboxyl terminus of phage HK022 Nun includes a novel zinc-binding motif and a tryptophan required for transcription termination

¹ Department of Biochemistry and Molecular Biophysics and ² Institute of Cancer Research College of Physicians and Surgeons, Columbia University, New York, NY 10032 USA; ³ Department of Chemistry, Swarthmore College, Swarthmore, Pennsylvania 19081 USA

The amino-terminal arginine-rich motif of the phage HK022 Nun protein binds phage  nascent mRNA transcripts while the carboxy-terminal domain binds RNA polymerase and arrests transcription. The role of specific residues in the carboxy-terminal domain in transcription termination were investigated by mutagenesis, in vitro and in vivo functional assays, and NMR spectroscopy. Coordination of zinc to three histidine residues in the carboxy-terminus inhibited RNA binding by the amino-terminal domain; however, only two of these histidines were required for transcription arrest. These results suggest that additional zinc-coordinating residues are supplied by RNA polymerase in the context of the Nun-RNA polymerase complex. Substitution of the penultimate carboxy-terminal tryptophan residue with alanine or leucine blocks transcription arrest, whereas a tyrosine substitution is innocuous. Wild-type Nun fails to arrest transcription on single-stranded templates. These results suggest that Nun inhibition of transcription elongation is due in part to interactions between the carboxy-terminal tryptophan of Nun and double-stranded DNA, possibly by intercalation. A model for the termination activity of Nun is developed on the basis of these data.

[Key Words: Nun protein; transcription termination; RNA binding; DNA binding; zinc-binding motif]