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

Abstract

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.

Figure 1

(A) Amino acid sequence of Nun protein and (B) RNA sequence of λ BOXB. (A) The ARM region is italicized, the carboxy-terminal 19-amino-acid peptide used for ¹H NMR spectroscopy is underlined, residues required for optimal Nun activity are in bold, and mutations known to have no effect on Nun activity are outlined. (B) Oligoribonucleotide that includes λ BOXB used in binding assays. The stem was elongated by an additional GC base pair to enhance stability.

Figure 2

Zn²⁺ titrations monitored by ¹H NMR spectroscopy. Regions of the NMR spectra containing the histidine imidazole resonances of the wild-type (A) and (B) H98A mutant 19-residue carboxy-terminal Nun peptide (B) are shown as functions of the indicated Zn²⁺ concentration. The wild-type peptide contains three histidine residues, H93, H98, and H100; the mutant peptide contains two histidine residues, H93 and H100. The wild-type peptide had a concentration of 220 μm and the mutant peptide had a concentration of 100 μm. (A) For the wild-type peptide in the absence of Zn²⁺, the histidine ε1 resonances have chemical shifts of 8.31 ppm (a), 8.05 ppm (b), and 8.00 ppm (c); the δ2 resonances have chemical shifts of 7.18 ppm (d), 7.08 ppm (e), and 7.04 ppm (f). (B) For the H98A mutant peptide in the absence of Zn²⁺, the histidine ε1 resonances have chemical shifts of 8.17 ppm (a) and 7.92 ppm (b); the δ2 resonances have chemical shifts of 7.12 ppm (c) and 7.00 ppm (d). For the wild-type peptide, the spectrum recorded at [Zn²⁺] = 660 μm was unchanged from the spectrum recorded at [Zn²⁺] = 330 μm; for the mutant peptide, the spectrum recorded at [Zn²⁺] = 330 μm was unchanged from the spectrum recorded at [Zn²⁺] = 165 μm (not shown). The individual resonances have not been specifically assigned to particular histidine residues in the Nun sequence. The unlabeled resonances in the spectra arise from the indole protons of W108.

Figure 3

Gel mobility shift assay. ³²P-labeled BOXB (20 nm) was incubated with wild-type, H93A, H98A, H100A, and H98C Nun (500 nm) in the presence and absence of 5 μm Zn²⁺. The reactions were subjected to electrophoresis on a 7.5% native polyacrylamide gel. The wild-type protein is inhibited from binding BOXB by Zn²⁺, whereas mutant proteins H93A, H98A and H100A are not.

Figure 4

In vitro transcription assays for wild-type, H93A, H98A, H100A, and H98C Nun proteins. The transcription complexes were allowed to proceed to the +15 site on the template by withholding UTP. The reaction was then allowed to proceed to the runoff product by the addition of 10 μm NTPs, in the presence or absence of Nun or Nun mutants. Nus factors were added to final concentrations of 5 μm to mimic in vivo conditions (B). The band identified as SA is a spontaneously arrested complex that is sensitive to GreB (Hung and Gottesman 1995). The mutant proteins were capable of arresting transcription only in the presence of the Nus factors in contrast to the wild-type protein, which arrests both in the presence and absence of the Nus factors. (NA) Nun-arrested transcript; (SA) spontaneous (Nun-independent) arrested transcripts; (RO) run-off transcript.

Figure 5

Effect of ZnCl₂ on Nun transcription arrest. Transcription reactions were performed essentially as described in Fig. 4, except that at the elongation step (after +15) ZnCl₂ was added, where indicated, at a concentration of 50 μm. Where indicated, Nun was added at 200 nm. The presence of ZnCl₂ increased the efficiency of arrest by wild-type Nun, but had no effect on the activity of the mutant proteins.

Figure 6

In vitro transcription. (A) In vitro transcription with RNAP in the absence of Nun (lane 1) and presence of wild-type (lane 2), W108A (lane 3), and H98AW108A (lane 4) Nun proteins in the absence of Nus factors (lanes 1–4). W108A Nun induces the production of a transcript approximately twice the length of the DNA template (SP) and does not arrest transcription at the sites where wild-type Nun does. H98AW108A Nun neither arrests transcription nor induces the production of the longer transcript. (Lanes 5–8) In vitro transcription with Nun proteins in the presence of the Nus factors. The Nus factors do not alter the activity of the mutant proteins. (B) Concentration dependence of W108A-induced template switching. Transcription was initiated with a template concentration of 5 nm and allowed to proceed to the +15 site on the template by withholding UTP; subsequently, the reaction was allowed to proceed to completion with the addition of 10 μm NTPs along with W108ANun. In this step, template concentration was supplemented to the levels indicated above the lanes. As template concentration was increased, the amount of the W108A-induced product was also increased. (RO) Runoff transcript; (SA) spontaneous (Nun-independent) arrest; (NA) Nun arrest; (SP) denotes switching product (W108A-induced transcript).

Figure 7

In vitro transcription of single-stranded (ss) and double-stranded (ds) templates. Transcription reactions were identical to those in Fig. 4 except that a doubled-stranded 110-bp template or a template with a 43-bp promoter and 77-nucleotide single-stranded region was used for the double-stranded and single-stranded transcription reactions, respectively. Where indicated, ZnCl₂ (50 μm) was added. (SA) Spontaneous (Nun-independent) arrest.

Figure 8

(A) Model of Nun termination/arrest. Nun first binds to RNAP and coordinates a zinc ion(s) in conjunction with the zinc finger of the β′ subunit of RNAP. H98 and either H93 or H100 displace the two water molecules. The basic residues carboxy-terminal to the histidine residues then position W108 to interact with the DNA template and physically block translocation of RNAP. (B) Proposed model of Nun–RNAP zinc-binding interaction: H98 and H93/H100 displace water molecules coordinated to Zn²⁺ in β′ amino-terminal zinc finger.