Biological Activities of Lasso Peptides and Structure–Activity Relationships - Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds - page 50

MccJ25 and capistruin are two antimicrobial lasso peptides produced by pro-
teobacteria that inhibit bacterial RNA polymerase (RNAP; Delgado et al. 2001 ;
Mukhopadhyay et al. 2004 ; Kuznedelov et al. 2011 ). RNAP is a nucleotidyl trans-
ferase enzyme involved in the transcription of the genetic information, i.e. RNA
synthesis from a DNA template, in all living cells (Cramer 2002 ; Borukhov and
Nudler 2008 ). While eukaryotes have three RNAPs involved in the synthesis of
ribosomal RNA, pre-messenger RNA and small RNAs (including transfer RNAs),
respectively, bacteria and archaea have one RNAP only. Bacterial RNAP is a large
protein (about 400 kDa). The core enzyme is constituted of five subunits (α 2 ββ′ω;
Borukhov and Nudler 2008 ). Its three-dimensional structure, obtained for the bacte-
ria Thermus aquaticus (Zhang et al. 1999 ), resembles a “crab claw”. Its active cen-
tre is located in the cleft between the two “pincers of the claw”, constituted by the β
and β′ subunits. It contains a Mg 2+ ion coordinated through three conserved aspar-
tate residues. The nucleoside triphosphate (NTP) substrates access the active centre
through the secondary channel (Vassylyev et al. 2007 ), and nascent RNA goes out
through the RNA exit channel. The core enzyme binds to one of a variety of initia-
tion factors (σ), involved in the recognition of promoter regions of DNA, to form
the RNAP holoenzyme (Vassylyev et al. 2002 ). The mechanism of transcription
consists of several key stages: (1) RNAP binding to the promoter to yield an RNAP/
promoter closed complex; (2) melting of a segment of promoter DNA next to the
transcription start site to yield the RNAP/promoter open complex; (3) abortive ini-
tiation, which consists of multiple rounds of synthesis and release of short (< 10 nt)
RNA products; (4) from 9- to 11-nt incorporation, release of the initiation factor
and processive elongation, through translocation of RNAP along the DNA tem-
plate; (5) termination: dissociation of the transcribing complex, when a termination
factor or signal is encountered. These steps rely on a complex set of interactions,
conformational changes and movements that are reviewed in Borukhov and Nudler
( 2008 ) and Svetlov and Nudler ( 2009 ). Bacterial RNAP constitutes an important
target for antibiotics, because it is essential for bacterial growth and survival, is well
conserved within bacteria and possesses particular features that permit targeting it
selectively without affecting eukaryotic RNAPs (Artsimovitch and Vassylyev 2006 ;
Chopra 2007 ; Mariani and Maffioli 2009 ; Srivastava et al. 2011 ).
Several potent broad-spectrum antibiotics target bacterial RNAP (Artsimovitch
and Vassylyev 2006 ; Mariani and Maffioli 2009 ; Srivastava et al. 2011 ; Fig. 3.6a ).
The best known are rifamycins and derivatives (Floss and Yu 2005 ), which belong
to the family of ansamycin antibiotics, characterized by an aromatic moiety bridged
at nonadjacent positions by an aliphatic chain. The rifamycins, isolated from an
actinomycete, display a broad-spectrum antibiotic activity against Gram-positive
and, to a lesser extent, Gram-negative bacteria. A rifamycin analogue, rifampicin, is
one of the main molecules used clinically for the treatment of tuberculosis, leprosy
and AIDS-associated mycobacterial infections (Floss and Yu 2005 ). The structure
of the Thermus aquaticus core enzyme, in complex with rifampicin (Campbell et al.
2001 ), has permitted to show that the antibiotic binds to a site of the β subunit lo-
cated in the path of nascent RNA. The different inhibitors of bacterial RNAP show
a wide diversity of structures, binding sites and mechanism of action (Fig. 3.6a ).