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

Abstract

The bacterial NusA protein enhances transcriptional pausing and termination and is known to play an essential role in antitermination. Antitermination is signaled by a nut-like cis-acting RNA sequence comprising boxB, boxA, and boxC. In the present study, we demonstrate a direct, specific high-affinity interaction between the rrn leader nut-like sites and the NusA proteins of Mycobacterium tuberculosis and Escherichia coli. This NusA-RNA interaction relies on the conserved region downstream of boxA, the boxC region, thus demonstrating a key function of this element. We have established an in vivo assay for antitermination in mycobacteria and use this to show that the M. tuberculosis rrn nut-like site enhances transcriptional read-through of untranslated RNA consistent with an antitermination signal within this site. Finally, we present evidence that this NusA-RNA interaction affects transcriptional events further downstream.N usA (N-using substance A) is a highly conserved transcription factor involved in transcriptional pausing, termination, and antitermination (reviewed in ref. 1). The protein is also thought to be essential for the increased elongation rate associated with rrn transcription and to be a constituent of the rrn antitermination complex (2, 3).The rrn antitermination complex ensures that transcription of rRNA is not terminated by Rho despite the nascent transcripts being untranslated and thus naked (4). The complex is believed to include RNA polymerase (RNAP), Nus factors A, B, E, and G, ribosomal protein S4, and possibly one or more additional cellular components (5-10). The antitermination complex is assembled after transcription of a nut-like site (hereafter referred to as nut site for brevity) consisting of boxB, boxA, and boxC within the rrn leader region (1, 4). However, it has been shown that boxA is sufficient to obtain antitermination (7, 11). It is not entirely clear how the assembly proceeds, but evidence suggests that binding of a NusB͞NusE heterodimer to boxA RNA may play a part, whereas the exact roles of NusA, boxB, and boxC remain unclear (6,7,11,12).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 (13-15). 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). Structural evidence suggests that KH domains interact sequence-specifically with four to five consecutive nucleotides in single-stranded regions (17)(18)(19)(20). Comparable structures of S1 domains in complex with nucleic acids are not available. Based on the NusA structure of Thermotoga maritima, Worbs et al. (15) proposed that the three RNA-binding domains in the NusA protein act together to form an ''extended RNA binding surface'' consistent with the ability to bind RNA with high affinity and specificity. NusA interacts with boxAB (21) as well as with mRNA (22). Although bacterial NusA proteins display a high degree of co...

Evolutionary Comparison of Ribosomal Operon Antitermination Function

“…However, because the use of such a system appears to be so widely conserved, the factors and mechanisms involved are important to understand in the context of overall rRNA expression and what the majority of RNA polymerase molecules are doing in the cell under fast growth conditions. To date, in addition to E. coli, M. tuberculosis and B. subtilis are the only other microorganisms for which this aspect of rRNA synthesis has been studied in any detail (3,16,17).…”

“…For example, examination of rrn leader and spacer regions from widely differing bacteria in the general location of the E. coli AT sequences reveals that AT features are readily recognized on the basis of sequence elements alone. Thus, although only E. coli and Mycobacterium tuberculosis AT sites have been studied and shown to have an antitermination function (3,4,50), it is likely that antitermination in rrn operons is a wellconserved mechanism.Studies of AT elements in rrn operons of E. coli have shown that boxA is necessary and sufficient for the antitermination function and that changes either in the boxA sequence itself or in Nus factors lead to a decreased or entire lack of AT activity (4,24,37,38,41,44,52). The boxA sequence occurs twice in E. coli rrn operons, and the two sequences differ by 1 base.…”

“…For example, examination of rrn leader and spacer regions from widely differing bacteria in the general location of the E. coli AT sequences reveals that AT features are readily recognized on the basis of sequence elements alone. Thus, although only E. coli and Mycobacterium tuberculosis AT sites have been studied and shown to have an antitermination function (3,4,50), it is likely that antitermination in rrn operons is a wellconserved mechanism.…”

“…The boxC region is a GT-rich region that is conserved in the lambda N antitermination and rrn AT systems (19). A crucial function for the boxC region is clear from studies of AT in rRNA operons of Mycobacterium tuberculosis, in which NusA binding to the AT sequence depends on boxA as well as sequences downstream of boxA, i.e., the boxC region (3,5). However, the boxC region can be eliminated from the E. coli test system with no detectable effect on terminator readthrough (4,44).…”

Essential Biological Processes of an Emerging Pathogen: DNA Replication, Transcription, and Cell Division in Acinetobacter spp

“…The boxA sequences are highly conserved across the eubacteria (consensus, 5Ј-GCUCUUU[A /G][A/G]A; Acinetobacter consensus, 5Ј-GAUCAUUAAG) and are integral to the formation of the NusB-NusE heterodimer that binds RNA polymerase (93). More recently it has been shown that NusA binds boxC (consensus, 5Ј-UGUG U[U/G][U/G]; Acinetobacter consensus, 5Ј-UGUGUGG), which is involved in antitermination complex formation (10,23). Targeting these protein-nucleic acid interactions may also be a valid approach for the development of new antibiotics, as preventing antitermination complex formation would severely disrupt cell proliferation.…”