1. Volume 1, Issue 2, Pages 265-275 (January 1998)
Independent Ligand-Induced Folding of the RNA-Binding Domain and Two Functionally Distinct Antitermination Regions in the Phage λ N Protein 
Jeremy Mogridge, Pascale Legault, Joyce Li, Mark D Van Oene, Lewis E Kay, Jack Greenblatt 
Molecular Cell 
Volume 1, Issue 2, Pages (January 1998)
DOI: /S (00)

2. Figure 1 Antitermination by the Phage λ N Protein
(A) Partial genetic and transcriptional map of bacteriophage λ.
(B) Modification of RNAP by N involves the formation of a ribonucleoprotein complex containing nut site RNA (boxA + boxB) and E. coli Nus factors.
Molecular Cell 1998 1, DOI: ( /S (00) )

3. Figure 2 Effect of N Binding on the boxB RNA Structure
1-D NMR spectra of the imino proton region of (A) free N1–22, (B) free boxB RNA, and boxB RNA bound to (C) N1–22, (D) N1–47, and (E) the full-length N protein. Sample concentrations were: (A) 2.5 mM 15N/13C N1–22; (B) 1.2 mM boxB RNA; (C) 3.0 mM 1:1 15N N1–22/boxB RNA; (D) 0.9 mM 1:115N N1–47/boxB RNA; (E) 0.12 mM 1:1 15N N/boxB RNA. Imino proton assignments are shown according to the numbering of the boxB RNA used for the NMR studies. Two additional G-C base pairs (g1-c19 and g2-c18) were added to the 15 nucleotide nutL boxB RNA hairpin stem.
Molecular Cell 1998 1, DOI: ( /S (00) )

4. Figure 3
Upon Binding to boxB RNA, Only the Arginine-Rich Domain of the N Protein Adopts a Specific Fold
(1H, 15N) HSQC spectra of the N protein and N protein fragments in the absence and in the presence of boxB RNA. Sample concentrations were: (A) 0.25 mM 15N/13C N1–22; (B) 2.5 mM 1:1 15N N1–22/boxB RNA; (C) 0.25 mM 1:1 15N/13C N1–22/G8A boxB RNA (E) 0.9 mM 15N N1–47; (F) 0.9 mM 1:1 15N N1–47/boxB RNA; (G) 0.18 mM 15N N; (H) 0.12 mM 1:1 15N N/boxB RNA. Specific signals are represented as follows: backbone amides (red); side chains (black); tryptophan indoles (arrows); arginine (R) side chain Hε-Nε groups (hexagons), the glutamine (Q), and asparagine (N) side chains (circles); selected backbone amides (boxed). Specific assignments obtained for the N1–22 peptide bound to boxB RNA (in [B]) are reported on all spectra recorded on complexes (B, F, and H). The assignments of N22 side chain signals were verified for N1–47 and N on the basis of NOEs (not shown). Spectral regions indicated by oval dashed lines represent correlations from backbone amides of glycines (G) and other (O) residues outside the arginine-rich domain of N that are not perturbed by the addition of boxB RNA. The arginine Hε-Nε correlations are folded in the nitrogen dimension in all these spectra. The sequence of the N protein is shown in (D), and colored boxes identify the arginine-rich RNA-binding domain (red), the NusA-binding region (green), and
the core polymerase-binding region (yellow). Bold letters indicate residues for which 1H-15N correlations were used to probe the structure of N1–47 and the full N protein (see text).
Molecular Cell 1998 1, DOI: ( /S (00) )

5. Figure 4
N1–47 Reverses the NusA Effect without Supporting Maximal Levels of Antitermination
(A) N1–47 binds NusA. E. coli extract containing overexpressed NusA was passed over columns containing 0.5 mg/ml GST (lane 1), GST-N (lane 10), or various deletion mutants of N fused to GST (lanes 2–9). Bound proteins were eluted with 1 M NaCl, subjected to SDS–PAGE, and stained with Coomassie blue.
(B) N34–47 binds NusA. E. coli extract containing overexpressed NusA was passed over columns containing 2 mg/ml GST (lane 1), or various deletion mutants of N fused to GST (lanes 2–6). Bound proteins were eluted with 1 M NaCl, subjected to SDS–PAGE and stained with Coomassie blue. The bands below the NusA band are proteolytic fragments of NusA.
(C) N1–47 supports an N/NusA/nut site complex. Reactions containing 32P-labeled nut site RNA, NusA, N, and N deletion mutants (as indicated) were electrophoresed on nondenaturing gels, dried, and exposed to film.
(D) N1–47 supports an intermediate level of antitermination. Transcription reactions containing 25 nM RNAP, various concentrations of NusA, and 100 nM full-length N or 100 nM N1–47 (as indicated) were electrophoresed on 6 M urea 4% polyacrylamide gels, dried, and exposed to film.
(E) The carboxyl terminus of N is important for antitermination. Transcription reactions containing 25 nM RNAP, 100 nM NusA, and 100 nM full-length N or various deletion mutants of N (as indicated) were electrophoresed on 6 M urea 4% polyacrylamide gels, dried, and exposed to film. Positions of terminated and run-off transcripts are indicated.
Molecular Cell 1998 1, DOI: ( /S (00) )

6. Figure 6 Multiple Regions of N Can Bind RNAP
(A) Binding of NusA and RNAP to amino-terminal deletion mutants of N. NusA (lane 2) or RNAP (lane 10) was loaded onto 0.5 mg/ml GST, GST-N73–107, GST-N58–107, GST-N48–107, GST-N34–107, GST-N23–107, and GST-N columns (as indicated). Bound proteins were eluted with 1 M NaCl, and, with 1/2 of the load, subjected to SDS–PAGE and stained with Coomassie blue. Lane 1 contained molecular weight standards (200 kDa, kDa, 97.4 kDa, 66.2 kDa, and 45.0 kDa).
(B) Binding of RNAP to carboxy-terminal deletion mutants of N. RNAP was loaded onto 1 mg/ml GST and 0.5 mg/ml GST-N1–39, GST-N1–47, GST-N1–58, GST-N1–73, GST-N1–89, and GST-N columns (as indicated). Bound proteins were eluted with 1 M NaCl, subjected to SDS–PAGE and stained with Coomassie blue. Lane 1 contained molecular weight standards (200 kDa, kDa, 97.4 kDa, 66.2 kDa, and 45.0 kDa).
(C) Binding of RNAP to carboxy-terminal deletion mutants of N1–47. RNAP (lane 2) was loaded onto 1 mg/ml GST, GST-N23–47, GST-N34–47, GST-N73–107, and GST-N columns (as indicated). Bound proteins were eluted with 1 M NaCl, and, with 1/2 of the load, subjected to SDS–PAGE, and stained with Coomassie blue. Lane 1 contained molecular weight standards (200 kDa, kDa, 97.4 kDa, 66.2 kDa, and 45.0 kDa).
(D) N1–47 binds RNAP core and holoenzyme whereas N73–107 only binds RNAP core component. RNAP core (lane 1) and holoenzyme (lane 11) were loaded onto GST, GST-N1–47, and GST-N73–107 columns (as indicated). Bound proteins were eluted with 1 M NaCl, and, with 1/8 of the loads, subjected to SDS–PAGE and stained with Coomassie blue.
Molecular Cell 1998 1, DOI: ( /S (00) )

7. Figure 5
The Carboxy-Terminal Domain of N Binds RNAP and Is Required to Support Factor-Independent Antitermination
(A) Deletion of the carboxy-terminal 18 amino acids of N abolishes its ability to support factor-independent antitermination. Transcription reactions containing 25 nM RNAP and 500 nM N or carboxy-terminal deletion mutants of N were electrophoresed on 6 M urea 4% polyacrylamide gels, dried, and exposed to film.
(B) N73–107 binds the RNAP core component. NusA (lane 1) or the core component of RNAP (lane 6) was loaded onto 2 mg/ml GST, GST-N34–47, GST-N73–107, and GST-N columns (as indicated). Bound proteins were eluted with 1 M NaCl, and, with 1/4 of the loads, subjected to SDS–PAGE and stained with Coomassie blue.
(C) Deletion of amino acids 74–107 of N impairs its ability to form a core antitermination complex. Reactions containing 32P-labeled nut site RNA, 25 nM RNAP core component, 100 nM NusA, and 500 nM N or N1–73 (as indicated) were electrophoresed on nondenaturing gels, dried, and exposed to film.
Molecular Cell 1998 1, DOI: ( /S (00) )

8. Figure 7
Summary of the Results of the Deletion Analysis on N, Including a Model of the Domain Structure of the λ N Protein
See text for further details.
Molecular Cell 1998 1, DOI: ( /S (00) )