Regulatory sRNAs in Cyanobacteria

As the transcriptional and post-transcriptional regulators of gene expression, small RNAs (sRNAs) play important roles in every domain of life in organisms. It has been discovered gradually that bacteria possess multiple means of gene regulation using RNAs. They have been continuously used as model organisms for photosynthesis, metabolism, biotechnology, evolution, and nitrogen fixation for many decades. Cyanobacteria, one of the most ancient life forms, constitute all kinds of photoautotrophic bacteria and exist in almost any environment on this planet. It is believed that a complex RNA-based regulatory mechanism functions in cyanobacteria to help them adapt to changes and stresses in diverse environments. Although lagging far behind other model microorganisms, such as yeast and Escherichia coli, more and more non-coding regulatory sRNAs have been recognized in cyanobacteria during the past decades. In this article, by focusing on cyanobacterial sRNAs, the approaches for detection and targeting of sRNAs will be summarized, four major mechanisms and regulatory functions will be generalized, eight types of cis-encoded sRNA and four types of trans-encoded sRNAs will be reviewed in detail, and their possible physiological functions will be further discussed

The Discovery and Characterization of NAD-Linked RNA

Over the past few decades, RNA has emerged as much more than just an intermediary in biology’s central dogma. RNA is now known to play a variety of catalytic, regulatory and defensive roles in living systems as demonstrated through the discoveries of ribozymes, riboswitches, microRNAs, small interfering RNAs, Piwi-interacting RNAs, small nuclear RNAs, clusters of regularly interspaced short palindromic repeat RNAs and long non-coding RNAs. In contrast to the functional diversity of RNA, the chemical diversity has remained primarily limited to canonical polyribonucleotides, the 5’ cap on mRNAs in eukaryotes, modified nucleotides and 3’-aminoacylated tRNAs. This disparity coupled with the powerful functional properties of small molecule-nucleic acid conjugates led us to speculate that novel small molecule-RNA conjugates existed in modern cells, either as evolutionary fossils or as RNAs whose functions are enabled by the small molecule moieties. We developed and applied a nuclease-based screen coupled with high-resolution liquid chromatography/mass spectrometry analysis to detect novel small molecule-RNA conjugates, broadly and sensitively. We discovered NAD-linked RNA in two types of bacteria and further characterized the small molecule and RNA in Escherichia coli. The NAD modification is found on the 5’ end of RNAs between 30 and 120 nucleotides long, and is surprisingly abundant at around 3,000 copies per cell. Subsequent experiments to characterize further NAD-linked RNA have been undertaken, including sequencing the RNAs to which NAD is attached and elucidating the biological functions of the small molecule-RNA conjugate. The development and application of a screen to detect novel nucleotide modifications that is independent of structure or biological context has the potential to increase our understanding of the functional and chemical diversity of RNA. The discovery and biological characterization of NAD-linked RNA can provide new examples of RNA biology and offer insight into the RNA world

Identification and characterisation of endogenous inducible promoters in Mycobacterium tuberculosis

PhDMycobacterium tuberculosis is one of the world’s most devastating pathogens. Despite completion of the genome sequence in 1998, research progress has been hampered by a lack of genetic tools and the difficulty of working with the organism. Existing genetic systems are limited by their lack of tight regulation or genetic instability. The aim of this study was to characterise and utilise a range of promoters to express mycobacterial genes in a controllable fashion by generating knockdown strains of a number of target genes, using both sense and antisense approaches. This would help to elucidate the function of a particular gene of interest and identify or validate new drug targets. Sets of genes shown to be inducible by certain stimuli such as tetracycline (Rv0277c, Rv0608, Rv0748, Rv1015c, Rv2487c and Rv3898c), streptomycin (whiB7), sodium dodecyl sulphate or ethanol (whiB6), hypoxia, nitric oxide and stationary phase (Rv2625c, Rv2626c, Rv2627c and hspX), or salicylate (Rv0560c) were selected from the literature. The upstream region of each gene was cloned in front of a reporter gene and activity was tested in M. smegmatis and/or M. tuberculosis. No inducible promoter activity was found for the upstream regions of the tetracycline, streptomycin, sodium dodecyl sulphate or ethanolresponsive genes. Inducible promoter activity was found for some of the hypoxiaresponsive genes and was monitored in relation to growth phase in M. tuberculosis wild type, a dosR deletion mutant and a Rv2625c deletion mutant. The upstream region of Rv0560c was found to contain a salicylateinducible promoter. Promoter elements of this promoter were identified and characterised in M. tuberculosis. Attempts to use the most promising promoters in an antisense setting using the reporter gene lacZ or the mycobacterial gene rpoB were unsuccessful

Structural studies of trans-translation

Ribosomes translate messenger RNA (mRNA) into protein in all living cells. The faultless production of protein is critical for a vast array of catalytic and structural roles and is essential for the survival of the cell. Ribosomes themselves are made up of both RNA and protein, and are composed of two subunits, each with a separate function. The small subunit reads the mRNA message, directing the large subunit to synthesize a sequence of amino acids to form a protein. In many cases, mRNA may be damaged or truncated in such a way that ribosomes reach the end of the message and become trapped. Rescuing stalled ribosomes is essential as an otherwise lethal build-up of unproductive ribosomes diminishes the translation capacity of a cell. This study focuses on an essential pathway called trans-translation, which resolves stalled ribosomes in nearly all bacteria. Two factors, transfer-messenger RNA (tmRNA) and small protein B (SmpB), form a complex that rescues the ribosome by terminating translation and releasing the ribosome from the mRNA message. In vitro biochemistry in conjunction with cryo-electron microscopy (cryo-EM) was used to visualize frozen snapshots of the ribosome undergoing trans-translation. The structures reveal the coordinated movement of tmRNA and SmpB through the ribosome. Binding interactions between tmRNA-SmpB and the ribosome explain why trans-translation only begins on ribosomes that reach the end of an mRNA and not for actively translation ones. SmpB plays an essential role in positioning tmRNA as together they mimic both a tRNA and mRNA. The movement of tmRNA-SmpB results in a stepwise message swapping from the original mRNA to tmRNA, facilitating the rescue of stalled ribosomes. Overall, this structural study advances our atomic level understanding of the mechanism of trans-translation.This degree was supported by a Gates Cambridge Scholarship

“Role of the two ClpP protease subunits in Mycobacterium tuberculosis”

PhDCaseinolytic (Clp) proteases are the most widespread energy-dependent proteases in bacteria. They are involved in protein quality control by degrading misfolded and aggregated proteins and have a role in regulatory proteolysis. The main group of substrates of the Clp proteases is the SsrA-tagged proteins, which arise in the presence of defective translation. SsrA tagging is carried out by tmRNA, encoded by ssrA, together with a protein partner SmpB. While most organisms have only one ClpP, Mycobacterium tuberculosis has two ClpP protease subunits (ClpP1 and ClpP2) with at least one of them essential for growth. Co-expression of clpP1 and clpP2 was demonstrated showing that clpP1 and clpP2 are not expressed under different conditions. The promoter region of clpP1P2 was identified, together with the potential ClgR binding site. A reporter system to assay ClpP1 and ClpP2 enzymatic activities was developed based on LacZ incorporating SsrA tag sequences. This showed that both ClpP1 and ClpP2 degrade SsrA-tagged LacZ, whilst only ClpP2 degrades untagged proteins. This suggests different pattern recognition for the two ClpP proteins with substrate recognition by ClpP1 dependent on the last three residues of the C-terminus of the tag sequence. Mutagenesis analysis of the accessory components demonstrated that ssrA is essential but SmpB deletion is viable. SmpB is not required for aerobic growth but the smpBΔ mutant strain was more sensitive to antibiotics targeting the ribosome as compared to wildtype cells