The α subunit of <i>E. coli</i> RNA polymerase activates RNA binding by NusA

Mah, Thien Fah; Kuznedelov, Konstantin; Mushegian, Arcady; Severinov, Konstantin; Greenblatt, Jack

doi:10.1101/gad.822900

Cited by 75 publications

(89 citation statements)

References 55 publications

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“…In line with this explanation, it is possible that certain, as-yet-unidentified features are shared between the probes that lead to complex formation with the E. coli NusA416. Alternatively, the lack of selectivity may be a consequence of the C-terminal truncation, which could modify the specificity of the protein either directly or indirectly by removing the dependence of accessory factors known to interact with the C-terminal domain, e.g., RNAP␣ or N (14,21).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

Section: A Specific Interaction Betweenmentioning

confidence: 99%

“…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). The protein was expressed and purified by using similar methodology to that used for the M. tuberculosis proteins and used in EMSAs.…”

Section: A Specific Interaction Betweenmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Arnvig¹,

Pennell²,

Gopal³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionmentioning

confidence: 99%

Section: A Specific Interaction Betweenmentioning

confidence: 99%

Section: A Specific Interaction Betweenmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Arnvig¹,

Pennell²,

Gopal³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…12,13 E. coli NusA also contains C-terminal auto-inhibitory (AR) domains which prevent the binding of RNA to S1/KH domains of NusA, and the inhibition is released after interaction of the AR domains with the α subunit C-terminal domain of RNAP. 14 Traditionally, there has always been a clear distinction between transcription initiation and elongation complexes. During the transition from initiation to elongation, the σ factor dissociates from RNAP followed by the association of NusA to regulate elongation and termination.…”

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confidence: 99%