The carboxyl terminus of phage HK022 Nun includes a novel zinc-binding motif and a tryptophan required for transcription termination.
The amino-terminal arginine-rich motif of the phage HK022 Nun protein binds phage lambda 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.
Watnick
R S
RS
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032 USA.
Herring
S C
SC
Palmer
A G
AG
Gottesman
M E
ME
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000737:chemistry*; D001483:Base Sequence; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011485:Protein Binding; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*; D014364:Tryptophan / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D015032:Zinc / Q000378:metabolism*
D017931:DNA Primers; D004277:DNA, Single-Stranded; C073501:Nun protein, Enterobacteria phage HK022; D014157:Transcription Factors; D014764:Viral Proteins; D006639:Histidine; D014364:Tryptophan; D015032:Zinc