Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors.

Mutations conferring resistance to translation inhibitors often alter the structure of rRNA. Reduced susceptibility to distinct structural antibiotic classes may, therefore, emerge when a common ribosomal binding site is perturbed, which significantly reduces the clinical utility of these agents. The translation inhibitors negamycin and tetracycline interfere with tRNA binding to the aminoacyl-tRNA site on the small 30S ribosomal subunit.

Structural Insights Lead to a Negamycin Analogue with Improved Antimicrobial Activity against Gram-Negative Pathogens.

Negamycin is a natural product with antibacterial activity against a broad range of Gram-negative pathogens. Recent revelation of its ribosomal binding site and mode of inhibition has reinvigorated efforts to identify improved analogues with clinical potential. Translation-inhibitory potency and antimicrobial activity upon modification of different moieties of negamycin were in line with its observed ribosomal binding conformation, reaffirming stringent structural requirements for activity.

Staphylococcus epidermidis Csm1 is a 3'-5' exonuclease.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) offer an adaptive immune system that protects bacteria and archaea from nucleic acid invaders through an RNA-mediated nucleic acid cleavage mechanism. Our knowledge of nucleic acid cleavage mechanisms is limited to three examples of widely different ribonucleoprotein particles that target either DNA or RNA. Staphylococcus epidermidis belongs to the Type III-A CRISPR system and has been shown to interfere with invading DNA in vivo.

Structure of the Cmr2-Cmr3 subcomplex of the Cmr RNA silencing complex.

The Cmr complex is an RNA-guided effector complex that cleaves invader RNA in the prokaryotic immune response mediated by the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas system. Here, we report the crystal structure of a Cmr subcomplex containing Cmr2 (Cas10) and Cmr3 subunits at 2.8 Å resolution.

Structure of the Cmr2 subunit of the CRISPR-Cas RNA silencing complex.

Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro.

The RNA-binding domain of bacteriophage P22 N protein is highly mutable, and a single mutation relaxes specificity toward lambda.

Antitermination in bacteriophage P22, a lambdoid phage, uses the arginine-rich domain of the N protein to recognize boxB RNAs in the nut site of two regulated transcripts. Using an antitermination reporter system, we screened libraries in which each nonconserved residue in the RNA-binding domain of P22 N was randomized. Mutants were assayed for the ability to complement N-deficient virus and for antitermination with P22 boxB(left) and boxB(right) reporters.

Bacteriophage P22 antitermination boxB sequence requirements are complex and overlap with those of lambda.

Transcription antitermination in phages lambda and P22 uses N proteins that bind to similar boxB RNA hairpins in regulated transcripts. In contrast to the lambda N-boxB interaction, the P22 N-boxB interaction has not been extensively studied. A nuclear magnetic resonance structure of the P22 N peptide boxB(left) complex and limited mutagenesis have been reported but do not reveal a consensus sequence for boxB.