The alpha subunit of E. coli RNA polymerase activates RNA binding by NusA

Abstract

The Escherichia coli NusA protein modulates pausing, termination, and antitermination by associating with the transcribing RNA polymerase core enzyme. NusA can be covalently cross-linked to nascent RNA within a transcription complex, but does not bind RNA on its own. We have found that deletion of the 79 carboxy-terminal amino acids of the 495-amino-acid NusA protein allows NusA to bind RNA in gel mobility shift assays. The carboxy-terminal domain (CTD) of the alpha subunit of RNA polymerase, as well as the bacteriophage lambda N gene antiterminator protein, bind to carboxy-terminal regions of NusA and enable full-length NusA to bind RNA. Binding of NusA to RNA in the presence of alpha or N involves an amino-terminal S1 homology region that is otherwise inactive in full-length NusA. The interaction of the alpha-CTD with full-length NusA stimulates termination. N may prevent termination by inducing NusA to interact with N utilization (nut) site RNA rather than RNA near the 3' end of the nascent transcript. Sequence analysis showed that the alpha-CTD contains a modified helix-hairpin-helix motif (HhH), which is also conserved in the carboxy-terminal regions of some eubacterial NusA proteins. These HhH motifs may mediate protein-protein interactions in NusA and the alpha-CTD.

Figure 1

Binding of NusA to RNA in the presence of the RNA polymerase α subunit. (A) Addition of increasing amounts of α to a constant amount of NusA results in an increase in complex formation. Reactions containing ³²P-labeled nut-site RNA and various combinations of 14 μM NusA and 1.25, 2.5, 5, or 10 μM α (as indicated) were electrophoresed on 7.5% nondenaturing gels, dried, and exposed to film. (B) Addition of increasing amounts of NusA to a constant amount of α results in increased complex formation. Reactions containing ³²P-labeled nut-site RNA and various combinations of 9 μM α and 3.5, 7, or 14 μM NusA (as indicated) were electrophoresed on 7.5% nondenaturing gels, dried, and exposed to film. (C) α-CTD stimulates RNA binding by NusA. Reactions containing ³²P-labeled nut-site RNA and various combinations of 13 μM NusA or NusA (amino acids 1–416), 4.5 or 9 μM α, 4.5 or 9 μM α-CTD or 11 μM α-NTD (as indicated) were electrophoresed on 7.5% nondenaturing gels, dried, and exposed to film.

Figure 2

RNA binding by NusA in the presence of α is sequence-specific and sensitive to a mutation in the S1 domain of NusA. (A) RNA binding by NusA in the presence of α is prevented by a mutation in the boxA portion of the nut site. Reactions containing wild-type or mutant ³²P-labeled nut-site RNA (as indicated) and various combinations of 10 μM α and 14 μM NusA (as indicated) were electrophoresed on 7.5% nondenaturing gels, dried, and exposed to film. (B) RNA binding by NusA in the presence of α is prevented by a mutation in the S1 domain of NusA. Reactions containing ³²P-labeled nut-site RNA and various combinations of 14 μM NusA, 14 μM NusA (amino acids 1–416), or 12 μM NusA R199A and 11 μM α (as indicated) were electrophoresed on 7.5% nondenaturing gels, dried, and exposed to film.

Figure 3

Interaction of α with the carboxy-terminal region of NusA. (A) Carboxy-terminal truncation of NusA prevents the α–NusA interaction. A mixture of four His₆-tagged NusA proteins (lane 2) was passed over columns containing affigel (lane 3) or increasing amounts of affigel-coupled α (lanes 4–6). Bound proteins were eluted with buffer containing 1M NaCl, subjected to SDS-PAGE, and stained with silver. (B) The carboxy-terminal region of NusA interacts directly with α. Buffer containing α and 0.2 mg/ml insulin (lane 2) was passed over columns containing affigel (lane 3) or affigel-coupled NusA (2 mg/ml) (lane 6) or affigel-coupled regions of NusA (lanes 4 and 5) (as indicated). The concentrations of amino- and carboxy-terminal regions of NusA on the columns were adjusted so that each had the same molar concentration as the full-length NusA. Bound protein was eluted with buffer containing 1 M NaCl, subjected to SDS-PAGE, and stained with silver. (C) α-CTD interacts with the 192 carboxy-terminal amino acids of NusA. A mixture of α, α-CTD, and α-NTD (lane 2) was passed over columns containing GST (lane 3) or increasing amounts of GST–NusA (amino acids 303–495) (lanes 4–6). As a control, buffer alone was passed over a GST–NusA (amino acids 303–495) column (lane 7). Bound proteins were eluted with buffer containing 1 M NaCl, subjected to SDS-PAGE, and stained with silver. * indicates a degradation product of α, as identified by mass spectrometry. (D) Helix–hairpin–helix motifs in the carboxy-terminal domains of bacterial RNA polymerase α subunits and NusA proteins. Identifiers in SWISSPROT and PDB databases are shown where available. Numbers indicate the distance, in amino acid residues, from the amino terminus of the protein. Roman numerals indicate the repeated motifs in the same protein. Residues conserved in many families of HhH proteins are indicated by bold type. Within the consensus line, certain categories are indicated: bulky hydrophobic residues (F, I, L, M, V, W, and Y; U in the consensus line), small side chains (A, G, and S; O in the consensus line), negatively charged residues (D and E; = in the consensus line), and positively charged residues (K and R; outlined letters in the alignment). Residues that may participate in charge–charge interactions are boxed. Underlined letters in the E. coli AlkA mismatch repair glycosylase sequence indicate the amino acids whose side chains make contacts with the phosphate residues in the DNA backbone, either directly or by coordinating a metal ion. The known elements of secondary structure are indicated by H for helix and h for hairpin.

Figure 4

A carboxy-terminally truncated NusA binds specifically to nut-site RNA. (A) RNA binding by NusA (amino acids 1–416) is prevented by a mutation in the boxA portion of the nut site. Reactions containing wild-type or mutant ³²P-labeled nut-site RNA (as indicated) and 3.5, 7, or 14 μM NusA or NusA (amino acids 1–416) (as indicated) were electrophoresed on 7.5% nondenaturing gels, dried, and exposed to film. (B) RNA binding by NusA (amino acids 1–416) is stabilized by amino acids 1–137 and 348–415 of NusA. Reactions containing ³²P-labeled nut-site RNA and 10 μM NusA (amino acids 132–416), NusA (amino acids 132–495), NusA (amino acids 1–348), NusA (amino acids 1–416) or NusA (as indicated) were electrophoresed on 7.5% nondenaturing gels, dried, and exposed to film.

Figure 5

The λ N protein binds directly to the 192 carboxy-terminal amino acids of NusA. E. coli extract containing additional NusA (lane 1), NusA (amino acids 1–399) (lane 4), or NusA (amino acids 303–495) (lane 7) was passed over columns containing 2 mg/ml GST (lanes 2, 5, and 8) or 0.5 mg/ml GST–N (lanes 3, 6, and 9). Bound proteins were eluted with buffer containing 1M NaCl, subjected to SDS-PAGE, and stained with silver.

Figure 6

The carboxy-terminal region of NusA is not necessary for enhancement of termination by NusA if a mutant RNA polymerase lacking the α-CTD is used. In vitro transcription reactions with wild-type RNA polymerase or a mutant RNA polymerase lacking the carboxy-terminal domain of the α subunit were incubated with either no NusA, NusA 1–348 (Δ), or NusA 1–495 (WT). The bar graph is based on the average values from 4 different experiments, only one of which is shown here.

Figure 7

Model for NusA function in elongation, termination, and antitermination. See text for details.