Functional importance of regions in Escherichia coli elongation factor NusA that interact with RNA polymerase, the bacteriophage λ N protein and RNA

Abstract

SummaryThe association of the essential Escherichia coli protein NusA with RNA polymerase increases pausing and the ef®ciency of termination at intrinsic terminators. NusA is also part of the phage l N protein-modi®ed antitermination complex that functions to prevent transcriptional termination. We have investigated the structure of NusA using various deletion fragments of NusA in a variety of in vitro assays. Sequence and structural alignments have suggested that NusA has both S1 and KH homology regions that are thought to bind RNA. We show here that the portion of NusA containing the S1 and KH homology regions is important for NusA to enhance both termination and antitermination. There are two RNA polymerase-binding regions in NusA, one in the amino-terminal 137 amino acids and the other in the carboxy-terminal 264 amino acids; only the aminoterminal RNA polymerase-binding region provides a functional contact that enhances termination at an intrinsic terminator or antitermination by N. The carboxy-terminal region of NusA is also required for interaction with N and is important for the formation of an N±NusA±nut site or N±NusA±RNA polymerase±nut site complex; the instability of complexes lacking this carboxy-terminal region of NusA that binds N and RNA polymerase can be compensated for by the presence of the additional E. coli elongation factors, NusB, NusG and ribosomal protein S10.

A high-affinity interaction between NusA and the rrn nut site in Mycobacterium tuberculosis

“…The M. tuberculosis NusA protein is highly homologous to the E. coli NusA protein. One major difference between the two proteins is that the C-terminal domain, which in E. coli seems to mask the RNA-binding domain(s), is absent from the M. tuberculosis protein, which therefore should enable the M. tuberculosis protein to bind RNA without the need for accessory factors (13,14). To investigate whether this indeed was the case, we used EMSAs to identify putative interactions between M. tuberculosis NusA and the M. tuberculosis rrn leader region.…”

“…These results demonstrate that the M. tuberculosis NusA protein binds to the first half of the rrn leader in vitro with high affinity. Moreover, the binding does not require accessory factors such as RNAP␣ or N, which are required for RNA binding of the E. coli NusA protein (14,21).…”

“…To determine whether the NusA-RNA interaction is likewise conserved, we tested the E. coli NusA protein and the E. coli rrnG nut site (nucleotides ϩ1 to ϩ64) in our assay. The E. coli NusA protein, unlike the M. tuberculosis protein, contains a C-terminal domain, which masks the RNA-binding region (14). We therefore used a truncated version consisting of amino acids 1-416 of the E. coli protein, which still contains all three RNA-binding domains (21).…”

“…Bacterial NusA proteins harbor three RNA-binding domains: S1, K homology 1 (KH1), and KH2 as well as an N-terminal domain that interacts with the RNAP (13)(14)(15). KH domains, like ribosomal protein S1 domains, are found in a variety of nucleic acid-binding proteins, often in multiple copies within one protein (16).…”

“…Although bacterial NusA proteins display a high degree of conservation, there is variability in the C-terminal region. Thus, in addition to the four domains present in the Mycobacterium tuberculosis protein, the Escherichia coli protein has a C-terminal domain, which in the absence of accessory factors masks one or more of the RNA-binding domains (14,21).…”