Project description:This experiment was done to look at the transcriptional profile of bacteria upon whiB2 transcriptional depletion. RNA-seq of an inducible whiB2 CRISPRi strain grown with 0 or 500 ng/mL anhydrotetracycline, which turns on the CRISPRi machinery. Samples were collected after 2 days of treatment.
Project description:ChIP-seq of mycobacterium tuberculosis expressing a 3xFLAG-tagged N-terminal WhiB2 compared with a strain carrying an empty FLAG (not attached to a protein)
Project description:ChIP-seq of Mycobacterium tuberculosis strain with a C-terminal FLAG-tagged WhiA allele compared to a strain carrying a FLAG attached to no protein (empty)
Project description:This experiment was performed to transriptioally profile bacteria with a whiA conditional knockdown using CRISPR interference (CRISPRi). A conditional knockdown strain was treated with 0 or 500 ng/mL anhydrotetracycline (ATC) for 4 days, which turns on the CRISPRi machinery. RNA extraction was performed using Zymo RNA miniprep kit. Libraries were constructed using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) using 150 ng of input RNA. Libraries were amplified using 8 cycles on the thermocycler. Post amplification libraries were size selected ay 250-450 bp in length using SparQ beads. Libraries were validated using Bioanalyzer high sensitivity DNA analysis.
Project description:Mitochondrial gene expression uses a non-universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt-)tRNAMet mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt-tRNAMet to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt-tRNAMet to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilization of m5C34 mt-tRNAMet in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt-tRNAMet function. Together, our data reveal how modifications in mt-tRNAMet are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAMet to recognise the different codons encoding methionine. HEK293 cell lines expressing His-FLAG-tagged NSUN3 or the His-FLAG tag alone were crosslinked using UV or treated with 5-azacytidine and analysed by CRAC
Project description:Binding of transcription factors to DNA is mediated by the recognition of the chemical signatures of the DNA bases and the three-dimensional shape of the DNA molecule. The direct contribution of DNA shape to DNA-binding specificity has been difficult to assess, as DNA shape is a consequence of its sequence. Here, we teased apart these two modes of recognition in the context of Hox-DNA binding. We made a series of mutations in Hox residues that, in a co-crystal structure, only recognize DNA shape, and tested the effect on DNA binding preferences using SELEX-seq. Analysis of shape features of selected sequences revealed that these residues are both necessary and sufficient for selection of sequences with distinct shape features. We used statistical machine learning to show that the accuracy of binding specificity predictions improves by adding shape features to a model that only depends on sequence. We conclude that shape readout is a direct and critical component of binding site selection by Hox proteins. Three rounds of SELEX were performed on a series of Hox mutants as described in Slattery et al, Cell, 2011 (PMID 22153072) and Riley et al, Methods in molecular Biology, 2014 (PMID 25151169). Briefly, His-tagged Scr and Antp mutant proteins were incubated with a randomized 16mer oligonucleotide library, and bound DNA was amplified and sequenced as described (PMID 22153072, PMID 25151169).
Project description:The dimerization and binding of DNA by BldD is affected by its interaction with cyclic di-GMP. The D116A mutant of BldD is partially impaired in its biding of cyclic di-GMP. ChIP-Seq was carried out to determine the difference in the degree of DNA binding by the wild type and D116A mutated BldD in Streptomyces venezuelae.