Molecular Mechanism of Transcription Inhibition by Peptide Antibiotic Microcin J25

Adelman, Karen; Yuzenkova, Yulia; Porta, A. La; Zenkin, Nikolay; Lee, Jookyung; Lis, John T.; Borukhov, Sergei; Wang, Michelle D.; Severinov, Konstantin

doi:10.1016/j.molcel.2004.05.017

Cited by 157 publications

(144 citation statements)

References 36 publications

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“…Other single-molecule studies have found that transcriptional initiation involves scrunching of the DNA template until contacts with the promoter are released (5,6), and supplied evidence for an elongation-incompetent intermediate state preceding transcriptional termination (14). Steady improvements in single-molecule assays have allowed the effects of transcriptional co-factors and effectors to be studied as well, including σ-factor (18), Gre A and GreB (34), ppGpp (50), and microcin J25 (123).…”

Section: Resultsmentioning

confidence: 99%

“…Instead, the frequency of pausing was significantly enhanced. These data, taken together with results from crosslinking studies showing that microcin J25 occupies the NTP entry channel, suggest that microcin J25 acts to inhibit transcription by blocking access of NTPs to the active site (123). Finally, using an ultra-stable assay with basepair resolution and scoring the positions of pauses induced by limiting a single NTP species, the DNA sequence of the template was reconstructed from the motions of as few as four RNAP molecules (35).…”

Section: Off-pathway Eventsmentioning

confidence: 99%

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Single-Molecule Studies of RNA Polymerase: Motoring Along

Herbert

Greenleaf

Block

2008

Annu. Rev. Biochem.

187

141

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Single-molecule techniques have advanced our understanding of transcription by RNA polymerase. A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps have been employed to probe the many facets of the transcription cycle. These approaches supply fresh insights into the means by which RNA polymerase identifies a promoter; initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity. Most studies to date have been performed with bacterial RNA polymerase, but work is also being carried out with eukaryotic polymerase (Pol II) and single-subunit polymerases from bacteriophages. We discuss recent progress achieved by single-molecule studies, highlighting some of the unresolved questions and ongoing debates.

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Section: Resultsmentioning

confidence: 99%

Section: Off-pathway Eventsmentioning

confidence: 99%

Single-Molecule Studies of RNA Polymerase: Motoring Along

Herbert

Greenleaf

Block

2008

Annu. Rev. Biochem.

187

141

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“…The 21-residue mature MccJ25 (2.1 kDa) was isolated from culture supernatants [2]. It exhibits a side-chain-to-backbone cyclization involving Glu 8 and the N-terminal glycine, and adopts a lasso-structure in which the Cterminal end of the peptide is threaded into the cyclic backbone [3]. We have shown recently that the two-chain analogue t-MccJ25 (initially called MccJ25-L [2]), obtained by cleavage with thermolysin, retains the lasso-structure of MccJ25 but loses the Val 11 -Pro 16 hairpin-like structure targeted by the enzyme [4] (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

“…Indeed, MccJ25 was shown to inhibit transcription by targeting the β subunit of RNA polymerase [6,7]. The molecular mechanism involves MccJ25 binding and obstruction of the RNA polymerase secondary channel, which in turn prevents the correct positioning of NTP substrates [8,9]. Conversely, few studies have addressed MccJ25 recognition at bacterial membranes.…”

Section: Introductionmentioning

confidence: 99%

The iron–siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11–Pro16 β-hairpin region in the recognition mechanism

Destoumieux-Garzón

Duquesne

Péduzzi

et al. 2005

Biochemical Journal

121

109

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The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA − strain and by using isogenic strains mutated in genes encoding the protein complex respectively.

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“…NTPs have been observed bound in the catalytic site and secondary channel, but not in the main channel, and there is no obvious path for NTPs to move from the main channel to the active site (18)(19)(20)(21)(22)(23). These observations have led to the proposal that the secondary channel is the major, and perhaps only, pathway for NTPs to enter the active site (24)(25)(26)(27). Biochemical studies, however, indicate that there may be NTP binding site (s) located in the main channel of RNAP (10,16,17).…”

mentioning

confidence: 99%

Templated nucleoside triphosphate binding to a noncatalytic site on RNA polymerase regulates transcription

Kennedy

Erie²

2011

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

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The regulation of RNA synthesis by RNA polymerase (RNAP) is essential for proper gene expression. Crystal structures of RNAP reveal two channels: the main channel that contains the downstream DNA and a secondary channel that leads directly to the catalytic site. Although nucleoside triphosphates (NTPs) have been seen only in the catalytic site and the secondary channel in these structures, several models of transcription elongation, based on biochemical studies, propose that template-dependent binding of NTPs in the main channel regulates RNA synthesis. These models, however, remain controversial. We used transient state kinetics and a mutant of RNAP to investigate the role of the main channel in regulating nucleotide incorporation. Our data indicate that a NTP specific for the i þ 2 template position can bind to a noncatalytic site and increase the rate of RNA synthesis and that the NTP bound to this site can be shuttled directly into the catalytic site. We also identify fork loop 2, which lies across from the downstream DNA, as a functional component of this site. Taken together, our data support the existence of a noncatalytic template-specific NTP binding site in the main channel that is involved in the regulation of nucleotide incorporation. NTP binding to this site could promote high-fidelity processive synthesis under a variety of environmental conditions and allow DNA sequence-mediated regulatory signals to be communicated to the active site.T he central role of RNA polymerase (RNAP) in transcription is to catalyze the processive synthesis of the growing RNA transcript with high-fidelity and at reasonable rates. This process is regulated both intrinsically, by RNA and DNA sequence elements, and extrinsically, by nucleotide availability and proteins that interact with the elongating RNAP (1-10). These interactions modulate the conformations of the RNAP ternary elongation complexes (RNAP, DNA, and RNA), which, in turn, affect the rate and fidelity of nucleotide addition and the response of RNAP to regulatory signals. The rate of nucleotide incorporation and the recognition of pause and termination signals by RNAP can be greatly influenced by the sequence of downstream DNA, as well as by the RNA transcript. Subtle changes in the sequence of the downstream DNA can result in dramatic changes in the rates of nucleotide incorporation and the efficiencies of pausing and termination (3, 10-15). The mechanism(s) by which the downstream DNA regulates these processes remains a mystery; however, it has been suggested that nucleoside triphosphates (NTPs) may interact with the downstream DNA to modulate nucleotide incorporation (10,16,17).Crystal structures of prokaryotic and eukaryotic RNAPs reveal two channels: the main channel, which is filled with the downstream DNA, and the secondary channel, which is a negatively charged, funnel-shaped pore that leads from the surface of the enzyme to the active site. NTPs have been observed bound in the catalytic site and secondary channel, but not in the main channel, and there...

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Molecular Mechanism of Transcription Inhibition by Peptide Antibiotic Microcin J25

Cited by 157 publications

References 36 publications

Single-Molecule Studies of RNA Polymerase: Motoring Along

Single-Molecule Studies of RNA Polymerase: Motoring Along

The iron–siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11–Pro16 β-hairpin region in the recognition mechanism

Templated nucleoside triphosphate binding to a noncatalytic site on RNA polymerase regulates transcription

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