Structure of a Mycobacterium tuberculosis NusA-RNA complex

Abstract

NusA is a key regulator of bacterial transcriptional elongation, pausing, termination and antitermination, yet relatively little is known about the molecular basis of its activity in these fundamental processes. In Mycobacterium tuberculosis, NusA has been shown to bind with high affinity and specificity to BoxB-BoxA-BoxC antitermination sequences within the leader region of the single ribosomal RNA (rRNA) operon. We have determined high-resolution X-ray structures of a complex of NusA with two short oligo-ribonucleotides derived from the BoxC stem-loop motif and have characterised the interaction of NusA with a variety of RNAs derived from the antitermination region. These structures reveal the RNA bound in an extended conformation to a large interacting surface on both KH domains. Combining structural data with observed spectral and calorimetric changes, we now show that NusA binding destabilises secondary structure within rRNA antitermination sequences and propose a model where NusA functions as a chaperone for nascently forming RNA structures.

Figure 1

(A) Schematic representation of the M. tb rrn, indicating the position of the P1 and Pcl1 promoters and the location of the BoxA, BoxB and BoxC sequences in the leader and spacer regions. The RNA sequence corresponding to the entire leader rrn antitermination region is shown below together with the name, sequence and location of ribo-oligonucleotides used in this study. BoxB, BoxA and BoxC sequences are highlighted in bold. The sequence elements of the λ nut site are shown for comparison. (B, C) The results of ribonuclease protection assays. (B) The products of T1 and CV1 digestion of RNA43 separated by urea denaturing electrophoresis. The RNA has been digested with increasing concentrations of T1 and CV1 indicated above each track. The numbering is the same as in panel A (left). The secondary structure of RNA43 derived from nuclease protection assays combined with the prediction from the mfold algorithm version 3.1 is shown (right). (C) T1 digests of RNA43 (left) and RNA43–NusAΔNt (right) analysed by integration of the band intensities from a phosphorimaged gel. Arrows indicate the positions of protected and hypersensitive bases that result in a decrease or increase in the integrated band intensity.

Figure 2

Analysis of RNA secondary structure by thermal denaturation. (A–C) Near-UV CD spectra recorded at 5°C upper curve and 90°C lower curve of (A) RNA11, (B) BoxC-loop and (C) RNA43. The spectra are expressed as Δɛ per nucleotide. (D) Thermal denaturation profile (CD₂₆₉) of RNA43 (—), BoxC-loop (—) and RNA11 (—).

Figure 3

(A–C) NusAΔNt-induced UV absorbance and CD spectral changes. (A) The UV absorbance spectra of BoxC-loop (black) and BoxC-loop upon addition of NusAΔNt (grey) (upper set of curves); RNA11 (black) and RNA11 upon addition of NusAΔNt (grey) (lower set of curves); and NusAΔNt (black) (lowest curve). The spectra were recorded at an RNA concentration of 4 μM with a two-fold excess of protein added, where appropriate. The contribution of NusAΔNt to the bound spectra has been subtracted. (B) The near-UV CD spectra of 3 μM BoxC-loop (grey) and BoxC-loop plus 13 μM NusAΔNt (black). (C) The near-UV CD spectra of 4 μM RNA13 (grey) and RNA13 plus 9 μM NusAΔNt (black). The spectra are expressed in Δɛ per nucleotide. At this concentration, the contribution from NusAΔNt to the overall CD spectrum above 260 nm is minimal. (D, E) Titration of NusAΔNt with rrn antitermination sequences measured by ITC: (D) BoxC-loop and (E) RNA13. In panels D and E, the top section shows the thermogram and the bottom section shows the line of best fit to the data.

Figure 4

The structure of the NusAΔNt–RNA11 complex. (A) Cartoon representations of the complex. The left- and right-hand panels show the complex in the same orientation. In the left-hand panel, the protein is shown as a green ribbon and the 11-mer RNA is shown in a stick representation associated with the KH1 and KH2 domains. The right-hand panel shows a representation of the molecular surface of NusAΔNt, where the calculated electrostatic potential has been mapped onto the surface of the protein. Regions of electropositive potential are shown in blue and regions of electronegative potential are coloured red. The RNA is shown in a stick representation. (B) Stereo view of the F_o−F_c omit map around nucleotides Ade49 to Gua53 at 3σ contouring. (C) The arrangement of domains in NusAΔNt. The individual domains are coloured blue (S1), green (KH1) and yellow (KH2) and are shown in a ribbon representation. β-Strands and α-helices are labelled per domain and in sequential order.

Figure 5

A schematic representation of the RNA–protein contacts in the NusAΔNt–RNA11 complex. Bases represented in grey circles are stacked and hydrogen bonding interactions coloured red are mediated through backbone–base contacts.

Figure 6

Details of the interaction of the KH domains with RNA11. (A) Interaction of KH1 with nucleotides Ade42 to Ura46 in the α/β groove of helices α2, α3 and β1–β3 of the KH1 domain. (B) A view highlighting the protein–nucleic acid interactions around Ade44 and Ade45. The protein is shown as a green ribbon. The hydrogen bonds of Ade44 to the backbone are shown together with the interactions of the 2′-hydroxy group of Ade45. (C) Stereo view of the interactions of Ade50, Ura51 and Ade52 with the protein. The path of the RNA along the groove of α′2/α′3 and β′3 in KH2 is shown. (D) Highlights of the network of polar interactions around Ade50 to Ade52. The protein and RNA are represented as in panel B.

Figure 7

(A) Multiple sequence alignment of the βααβ motif of KH domains. Secondary structure elements were assigned based on the X-ray structure. Residues that are 100% conserved include the glycines of the GXXG motif and isoleucine in β-strand 3 where the backbone is in contact with Ade44 or Ade50. (B) Structural superposition of KH1 in the NusAΔNt–RNA11 complex (grey) with KH1 in the NusAΔNt–RNA12 complex (yellow) and with KH2 (blue). (C) Structural superposition of RNA in KH2 (blue) with RNA bound to Nova (red).