Design of novel peptide inhibitors against the conserved bacterial transcription terminator, Rho

Abstract

The transcription terminator Rho regulates many physiological processes in bacteria, such as antibiotic sensitivity, DNA repair, RNA remodeling, and so forth, and hence, is a potential antimicrobial target, which is unexplored. The bacteriophage P4 capsid protein, Psu, moonlights as a natural Rho antagonist. Here, we report the design of novel peptides based on the C-terminal region of Psu using phenotypic screening methods. The resultant 38-mer peptides, in addition to containing mutagenized Psu sequences, also contained plasmid sequences, fused to their C termini. Expression of these peptides inhibited the growth of Escherichia coli and specifically inhibited Rhodependent termination in vivo. Peptides 16 and 33 exhibited the best Rho-inhibitory properties in vivo. Direct high-affinity binding of these two peptides to Rho also inhibited the latter's RNA-dependent ATPase and transcription termination functions in vitro. These two peptides remained functional even if eight to ten amino acids were deleted from their C termini. In silico modeling and genetic and biochemical evidence revealed that these two peptides bind to the primary RNA-binding site of the Rho hexamer near its subunit interfaces. In addition, the gene expression profiles of these peptides and Psu overlapped significantly. These peptides also inhibited the growth of Mycobacteria and inhibited the activities of Rho proteins from Mycobacterium tuberculosis, Xanthomonas, Vibrio cholerae, and Salmonella enterica. Our results showed that these novel anti-Rho peptides mimic the Rho-inhibition function of the 42-kDa dimeric bacteriophage P4 capsid protein, Psu. We conclude that these peptides and their C-terminal deletion derivatives could provide a basis on which to design novel antimicrobial peptides.

Peptides designed from a bacteriophage capsid protein function as synthetic transcription repressors

“…1 B ). Earlier, we observed that up to eight amino acid deletions (peptide 33Δ8) from the CTD of peptide 33 did not affect its Rho-binding or cell growth inhibitory functions ( 10 ). The net charge of peptide 33 changes significantly upon CTD deletions, which in turn affects their functions.…”

“…Psu’s solvent-exposed C-terminal domain (CTD) (helix 6 and helix 7) interacts directly with Rho, and its N-terminal domain stabilizes its structure ( 9 ). We cloned the C-terminal helix-7 in a suitable expressing vector and randomly mutagenized it to select clones that inhibit the functions of the Rho protein (( 10 ), Fig. S1 A ).…”

“…S1 A ). Two of these clones expressing 45-mer peptides, peptide 16 and peptide 33, were characterized for their specific functions to inhibit Rho-dependent termination in vivo and in vitro by direct binding to the Rho protein (( 10 ), Fig. S1 , B and C ).…”

“…S1 , B and C ). Their expression also caused toxicity to E. coli, Mycobacterium smegmatis , and Mycobacterium bovis ( 10 ). These peptides could be used to further develop AMPs.…”

Widespread gene regulator Psu inhibits transcription termination factor ρ by forced hyper-oligomerization

“…While short peptides or lowmolecular-weight compounds will not support the dimerization of two open ρ rings akin to Psu, substances that bind to and increase the pitch of open ρ hexamers may conceivably allow expansion into inactive ρ filaments. Notably, Psu-derived, ρ-inhibiting peptides that are active in vivo have already been designed 75 , but the structural basis of their modes of action remains to be explored.…”