Antibiotic drugs targeting bacterial RNAs

AbstractRNAs have diverse structures that include bulges and internal loops able to form tertiary contacts or serve as ligand binding sites. The recent increase in structural and functional information related to RNAs has put them in the limelight as a drug target for small molecule therapy. In addition, the recognition of the marked difference between prokaryotic and eukaryotic rRNA has led to the development of antibiotics that specifically target bacterial rRNA, reduce protein translation and thereby inhibit bacterial growth. To facilitate the development of new antibiotics targeting RNA, we here review the literature concerning such antibiotics, mRNA, riboswitch and tRNA and the key methodologies used for their screening

Selection and characterization of RNA aptamers that detect a quaternary structure for ribosomal protein S7

Here we report on the selection and characterization of RNA aptamers that recognize E. coli ribosomal protein S7. Ribosomal protein S7 plays two important roles in ribosome biogenesis: (1) as an assembly initiator, S7 nucleates the folding of the 3\u27 major domain of 16S rRNA, and (2) it binds to the str operon and represses the translation of S12, S7, and EF-G. The primary and secondary structures of the S7 binding sites of rRNA and mRNA share limited sequence and structural homology and the required elements for high affinity binding have not been entirely elucidated. We have selected RNA aptamers that share very little primary sequence homology to either the S7 binding site of 16S rRNA or to the intercistronic region of str mRNA. Many of the aptamers are expected to fold into three-helix junctions, a structure particularly reminiscent of the mRNA. Interestingly, the aptamers exhibit cooperative binding with Hill coefficients of ~3 indicating that they are detecting a quaternary structure of S7. We have found that the S7 aptamers use the same amino acids and structural elements to bind S7 as the rRNA and mRNA indicating that the same binding site is used for all three RNAs. With gel filtration, we were only able to isolate the aptamer/S7 complex at a 1:1 stoichiometry, indicating that the proposed quaternary structure of S7 is weak. However, deletion of the β-ribbon nearly eliminates cooperative aptamer binding suggesting that this structural element may be involved in protein-protein interaction. Furthermore, pre-treatment of native S7 with the N-terminal extension also results in a significant reduction in cooperative aptamer binding. The results presented here suggest that S7 itself may undergo conformational rearrangement subsequent to 16S rRNA binding, and may help explain the strong temperature-dependent rearrangements at the binding site of S7 within the 16S rRNA. Furthermore, we propose that the weak, multimeric interaction of S7 may have a role in the retroregulation of S12. S7 may bind to the mRNA in a pre-multimerized form or multimerize subsequent to binding, resulting in ribosome stalling due to the multimeric obstacle. If the S7/S7 interaction is weak however, then it may be easily disrupted by repeated ribosome bombardment, causing eventual decay of the multimer and relieving some of the translational repression. Translational repression of the genes encoding S7 and EF-G would remain constant over time however, because the monomeric S7 bound more tightly to the intercistronic region would continue to prevent translational coupling with the upstream gene encoding S12

Nesprin-1 is required to maintain genomic stability and prevent tumorigenesis

Nuclear envelope (NE) proteins have fundamental roles in maintaining nuclear structure, cell signaling, chromatin organization and gene regulation, and mutations in genes encoding NE components were identified as primary cause of a number of age associated diseases and cancer. Nesprin-1 belongs to a family of multi-isomeric NE proteins that are characterized by spectrin repeats. Our results imply interactions between spectrin repeats and an interaction of Nesprin-1 with Nesprin-2. Furthermore, we analysed NE components in various tumor cell lines and found that Nesprin-1 levels were strongly reduced associated with alterations in further NE components. By reducing the amounts of Nesprin-1 by RNAi mediated knock down we could reproduce those alterations in mouse and human cell lines pointing towards a key role for Nesprin-1 in the maintenance of nucleus morphology, centrosome positioning, nuclear membrane structure, cytoskeleton organization, and cellular senescence. In a search for novel Nesprin-1 binding proteins we identified MSH2, MSH6, and DDB1 proteins of the DNA damage response pathway as interactors. We found alterations in the mismatch repair pathway in cells with lower Nesprin-1 levels. We also noticed an increased number of γH2AX foci in the absence of exogenous DNA damage as was seen in tumor cells. The levels of phosphorylated kinases Chk1 and 2 were altered in a manner resembling tumor cells and the levels of Ku70 were low and the protein was not recruited to the DNA after HU treatment. Our findings indicate a role for Nesprin-1 in the DNA damage response pathway and propose Nesprin-1 as novel regulator of tumorigenesis and genome instability. Loss of Nesprin-1 might play a significant role in cancer progression