RNA degradation is a central process required for transcriptional regulation. Eventually, this process degrades diribonucleotides into mononucleotides by specific diribonucleases. In Escherichia coli, oligoribonuclease (Orn) serves this function and is unique as the only essential exoribonuclease.
View Article and Find Full Text PDFThe nucleotide messenger (p)ppGpp allows bacteria to adapt to fluctuating environments by reprogramming the transcriptome. Despite its well-recognized role in gene regulation, (p)ppGpp is only known to directly affect transcription in Proteobacteria by binding to the RNA polymerase. Here, we reveal a different mechanism of gene regulation by (p)ppGpp in Firmicutes: (p)ppGpp directly binds to the transcription factor PurR to downregulate purine biosynthesis gene expression upon amino acid starvation.
View Article and Find Full Text PDFThe alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses. There is evidence for potential existence of a third alarmone, guanosine-5'-monophosphate-3'-diphosphate (pGpp), with less-clear functions. Here, we demonstrate the presence of pGpp in bacterial cells, and perform a comprehensive screening to identify proteins that interact respectively with pGpp, ppGpp and pppGpp in Bacillus species.
View Article and Find Full Text PDFDegradation of RNA polymers, an ubiquitous process in all cells, is catalyzed by specific subsets of endo- and exoribonucleases that together recycle RNA fragments into nucleotide monophosphate. In γ-proteobacteria, 3-'5' exoribonucleases comprise up to eight distinct enzymes. Among them, Oligoribonuclease (Orn) is unique as its activity is required for clearing short RNA fragments, which is important for cellular fitness.
View Article and Find Full Text PDFProtein interactions with nucleic acids are important for the synthesis, regulation, and stability of macromolecules. While a number of assays are available for interrogating these interactions, the differential radial capillary action of ligand assay (DRaCALA) has been developed as an easy and flexible platform that allows for the study of individual interactions when carrying out high-throughput screening for novel binding proteins and small molecule inhibitors. In this article, we describe the principle of DRaCALA and methods that utilize DRaCALA to determine the affinity and specificity of individual protein-nucleic acid interactions as well as uses for screening for binding proteins and chemical inhibitors.
View Article and Find Full Text PDFBis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that regulates processes, such as biofilm formation and virulence. During degradation, c-di-GMP is first linearized to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG) and subsequently hydrolyzed to two GMPs by a previously unknown enzyme, which was recently identified in as the 3'-to-5' exoribonuclease oligoribonuclease (Orn). Mutants of accumulated pGpG, which inhibited the linearization of c-di-GMP.
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