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Tie Me Up, Tie Me Down: Inhibiting RNA Polymerase

2008; Cell Press; Volume: 135; Issue: 2 Linguagem: Inglês

10.1016/j.cell.2008.09.052

1097-4172

Rui Sousa,

Bacteriophages and microbial interactions

Mechanistic understanding of antibiotic action can yield crucial insights that aid in the design of new antibiotics. In this issue, Mukhopadhyay et al., 2008Mukhopadhyay J. Das K. Ismail S. Koppstein D. Jang M. Hudson B. Sarafianos S. Tuske S. Patel J. Jansen R. et al.Cell. 2008; (this issue)PubMed Google Scholar uncover the mechanism by which the antibiotic myxopyronin inhibits bacterial RNA polymerase, suggesting a new target region in RNA polymerase for inhibitor design. Mechanistic understanding of antibiotic action can yield crucial insights that aid in the design of new antibiotics. In this issue, Mukhopadhyay et al., 2008Mukhopadhyay J. Das K. Ismail S. Koppstein D. Jang M. Hudson B. Sarafianos S. Tuske S. Patel J. Jansen R. et al.Cell. 2008; (this issue)PubMed Google Scholar uncover the mechanism by which the antibiotic myxopyronin inhibits bacterial RNA polymerase, suggesting a new target region in RNA polymerase for inhibitor design. Given the implications for human health, the development of antibiotic resistance in pathogenic bacteria is a topic of more than academic interest. The emergence of bacterial strains resistant to rifamycins, a group of antibiotics that inhibit the bacterial RNA polymerase (RNAP), has greatly compromised the treatment of tuberculosis. Rifamycins are the only antibiotics in widespread use to treat tuberculosis (Shinnick, 1996Shinnick T. Current Topics in Microbiology and Immunology. Springer-Verlag, New York1996Google Scholar), a disease that is estimated by the World Health Organization to have killed 1.7 million people in 2006 alone. Thus, there is an urgent need to develop new antibiotics to treat infections caused by rifamycin- or multidrug-resistant bacteria. The bacterial RNAP is an attractive target for development of such drugs (Chopra, 2007Chopra I. Curr. Opin. Investig. Drugs. 2007; 8: 600-607PubMed Google Scholar). As a large multisubunit enzyme, RNAP is expected to have multiple sites that could be bound by small molecules. Also, although the transcriptional machinery of prokaryotes and eukaryotes exhibits similarities in structure and mechanism, there has been sufficient divergence in even the most conserved RNAP subunits to make specific targeting of bacterial or eukaryotic RNAPs possible. Indeed, several antibiotics that specifically inhibit bacterial RNAP or eukaryotic RNAP II have been found to occur naturally; rifamycins are one such example (Campbell et al., 2001Campbell E.A. Korzheva N. Mustaev A. Murakami K. Nair S. Goldfarb A. Darst S.A. Cell. 2001; 104: 901-912Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar). It seems wise then to begin with what nature has developed in our search for improved antibiotic compounds. This is the approach taken by Mukhopadhyay et al., 2008Mukhopadhyay J. Das K. Ismail S. Koppstein D. Jang M. Hudson B. Sarafianos S. Tuske S. Patel J. Jansen R. et al.Cell. 2008; (this issue)PubMed Google Scholar in their study, in this issue of Cell, describing the mechanism of bacterial RNAP inhibition by the α-pyrone antibiotic myxopyronin (myx), which is produced by the bacteria Myxococcus fulvus (Irschik et al., 1983Irschik H. Gerth K. Hofle G. Kohl W. Reichenbach H. J. Antibiot. (Tokyo). 1983; 36: 1651-1658Crossref PubMed Scopus (99) Google Scholar). RNAP resembles a crab claw (Figure 1A) that must open to allow DNA to bind in the catalytic cleft. It does so via a swinging motion of one of the "pincers" (the clamp) that is formed by part of the largest RNAP subunit (β′ in bacterial RNAP; RPB1 in eukayotic RNAP II) (Figures 1B and 1C). Following DNA binding, the clamp swings back to close over the DNA (Figure 1D) in a step that appears coupled to promoter melting during transcription initiation. This step is also likely to be required for transcription complex stability and processivity during transcription elongation. To study how RNAP is inhibited by myx, Mukhopadhyay et al. needed to find the myx-binding site. A screen for myx resistance in the bacteria Escherichia coli uncovered mutations in three RNAP residues in the switch region that forms the hinge around which the clamp rotates to open and close the DNA-binding cleft. The authors then performed targeted mutagenesis of the RNAP switch region in E. coli to identify residues that, when mutated, confer myx resistance. In the 125 myx-resistant E. coli strains they identified, the authors found 13 residues within the switch region whose individual mutation conferred resistance to myx. Remarkably, all 13 of these residues are conserved in the RNAPs of different bacteria, thus explaining myx's broad spectrum activity against both Gram-positive and Gram-negative pathogens. Furthermore, consistent with myx's bacteria-specific inhibitory activity, 3 of these 13 residues are not conserved in eukaryotic RNAPs. Binding experiments showed that mutations in any of these 13 residues disrupted myx binding, thus implicating the switch region as the site of myx binding. Mukhopadhyay and colleagues confirmed this mode of myx binding by determining the structure of RNAP from the bacteria Thermus thermophilus in a complex with myx. The structure revealed that myx is almost completely buried in a hydrophobic pocket within the switch, far from the catalytic center (Figure 1E). This suggested that myx functions allosterically by inhibiting either the opening or closing of the clamp. But was myx jamming the clamp's hinge to prevent clamp opening and DNA binding, or was it binding to the opened clamp to prevent clamp closure after DNA has bound? Although the crystal structure implied the former scenario, it was possible that this aspect of the structure was misleading given that crystal packing can alter protein conformations from their state in solution. To distinguish between these two possibilities, Mukhopadhyay et al. characterized the effects of myx on RNAP promoter binding in vitro. They found that myx inhibited promoter binding only if it was added to the in vitro reaction containing RNAP before the promoter DNA was added. If myx was added after the promoter DNA was introduced into the reaction, no binding inhibition was observed. Moreover, myx had similar inhibitory effects on both abortive (short) and runoff (long) transcript synthesis in vitro, consistent with myx inhibiting DNA binding rather than affecting transcription complex stability or processivity following DNA binding. Furthermore, myx did not inhibit RNAP binding to promoters that lacked the DNA duplex region usually bound within the RNAP "claw" and melted during transcription initiation, implying that myx locks the clamp in a closed or partially closed state that still allows the RNAP to accommodate a single strand of DNA but not a duplex. This characterization of the mechanism of RNAP inhibition by myx has further implications for the mode of action of two other antibiotics, corallopyronin and ripostatin. Mukhopadhyay et al. showed that both of these antibiotics have identical patterns of inhibition and order-of-addition effects in promoter-binding experiments. Thus, it is likely that these antibiotics have a mechanism akin to myx—binding in the RNAP switch region to block clamp opening and promoter binding. Indeed, almost without exception, mutant E. coli resistant to any one of these three antibiotics are also resistant to the other two. This is particularly surprising for ripostatin, a macrocylic-lactone molecule that, unlike corallopyronin, is similar to myx only in terms of size and general hydrophobicity. Other compounds inhibit RNAP by interfering with conformational changes. For example, streptolydigin binds to bacterial RNAP near the base of the pincer opposite the clamp and is believed to function by inhibiting the conformational changes required to organize the RNAP catalytic center or to translocate the RNAP during each cycle of nucleotide addition (Tuske et al., 2005Tuske S. Sarafianos S.G. Wang X. Hudson B. Sineva E. Mukhopadhyay J. Birktoft J.J. Leroy O. Ismail S. Clark Jr., A.D. et al.Cell. 2005; 122: 541-552Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The toxin of the death cap mushroom, α-amanitin, binds to eukaryotic RNAP II in a region near the streptolydigin-binding site and has been proposed to work via a similar mechanism (Kaplan et al., 2008Kaplan C.D. Larsson K.M. Kornberg R.D. Mol. Cell. 2008; 30: 547-556Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Why might sites of focused conformational change be preferentially targeted by these naturally occurring inhibitors? Most obviously, jamming the moving parts of the transcription machine, like jamming the moving parts of any machine, is an effective way to stop it in its tracks. But there may be other features of these sites that make them ready targets for small-molecule inhibitors. As points of articulation between different RNAP domains or subdomains, these sites may be like the chinks in a suit of armor—weak points where a sword can be driven in. Indeed, myx is bound in the switch region in a manner that suggests it has wormed its way into this site, a mechanism that would require RNAP flexing to allow myx binding. This study is the latest chapter in a two decade long effort to understand RNAP structure and mechanism. With the myx-RNAP structure in hand, Mukhopadhyay et al. can now propose rational modifications that should enhance myx affinity and effectiveness in RNAP inhibition. This next step can only be contemplated because of the advance in understanding of myx mechanism achieved through a comprehensive approach that brought together genetics, biochemistry, and crystallography. Mechanistic understanding is the prerequisite to manipulating what nature has provided. To paraphrase Al Pacino in the film Scarface, when you get the knowledge, then you get the power. The RNA Polymerase "Switch Region" Is a Target for InhibitorsMukhopadhyay et al.CellOctober 17, 2008In BriefThe α-pyrone antibiotic myxopyronin (Myx) inhibits bacterial RNA polymerase (RNAP). Here, through a combination of genetic, biochemical, and structural approaches, we show that Myx interacts with the RNAP "switch region"—the hinge that mediates opening and closing of the RNAP active center cleft—to prevent interaction of RNAP with promoter DNA. We define the contacts between Myx and RNAP and the effects of Myx on RNAP conformation and propose that Myx functions by interfering with opening of the RNAP active-center cleft during transcription initiation. Full-Text PDF Open Archive