Biology subjects

Halada, P.

Publications and source records attributed to Halada, P..

5 recordsLinked to original sources

bioRxiv2024

Expanding the CarD interaction network: CrsL is a novel transcription factor in Mycobacterium smegmatis

Bacterial transcription regulation is critical for adaptation and survival. CarD is an essential transcription factor in mycobacteria involved in regulation of gene expression. We searched for CarD interaction partners in the model organism Mycobacterium smegmatis and identified two proteins: ApeB (MSMEG_5828) and an uncharacterized protein, which we named CrsL (MSMEG_5890). While ApeB interacted with CarD only when CarD was overexpressed, CrsL associated with CarD at its physiological levels. CrsL is a 5.7 kDa protein shown by NMR to be intrinsically disordered. CrsL homologs are present in actinobacteria including pathogenic species such as Mycobacterium tuberculosis. CrsL directly interacts with CarD and binds RNAP. ChIP-seq showed that CrsL associates with promoters of actively transcribed genes and [~]75 % of these regions are also associated with CarD. RNA-seq showed [~]50% and [~]66% overlap in differentially expressed genes between CrsL and CarD knockdowns during exponential and stationary phases, respectively. CrsL represses expression of DesA desaturase (MSMEG_5773) and DEAD/DEAH-box RNA helicase MSMEG_1930, which are important for adaptation to cold stress. Furthermore, CrsL promotes the growth of M. smegmatis at elevated temperature. In summary, this study identifies CrsL as a novel actinobacterial transcription factor and provides a basis for its further investigation.

microbiology↗

bioRxiv2024

HelD is a Global Transcription Factor Enhancing Gene Expression in Rapidly Growing Mycobacteria

HelD protein, also named HelR (encoded by MSMEG_2174 in Mycobacterium smegmatis), interacts with mycobacterial RNA polymerase (RNAP) and affects rifampicin resistance in Mycobacterium abscessus. Here, we provide data on rifampicin resistance and helD presence in the genomes of other clinically relevant nontuberculous mycobacteria. We show that helD is primarily found in rapidly growing mycobacteria, such as M. smegmatis, where we detected HelD at a subset of promoters that can also associate with CarD and RbpA. Transcriptome analysis of a helD deletion strain using RNA-seq revealed that HelD enhances gene expression during exponential growth and decreases it in stationary phase, during which we observed reduced levels of CarD, RbpA, and GTP, the initiation nucleotide for the majority of M. smegmatis transcripts. We propose a model in which HelD releases abortive RNAP complexes and confirm that HelD dissociates RNAP from the promoter in vitro. HelD not only helps mycobacteria overcome rifampicin treatment but also supports efficient transcription during rapid growth, which indicates a dual role of this transcription regulator.

microbiology↗

bioRxiv2023

Stem-loop induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control

ATF4 is a master transcriptional regulator of the integrated stress response leading cells towards adaptation or death. ATF4s induction under stress was thought to be mostly due to delayed translation reinitiation, where the reinitiation-permissive uORF1 plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations, but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrate that the canonical ATF4 translation start site is substantially leaky-scanned. Thus, ATF4s translational control is more complex than originally described underpinning its key role in diverse biological processes.

molecular biology↗

bioRxiv2020

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

RNA synthesis is central to life, and RNA polymerase depends on accessory factors for recovery from stalled states and adaption to environmental changes. Here we investigated the mechanism by which a helicase-like factor HelD recycles RNA polymerase. We report a cryo-EM structure of an unprecedented complex between the Mycobacterium smegmatis RNA polymerase and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNA polymerase channels that are responsible for DNA binding and substrate delivery to the active site, thereby locking RNA polymerase in an inactive state. We show that HelD prevents non-specific interactions between RNA polymerase and DNA and dissociates transcription elongation complexes, but does not inhibit RNA polymerase binding to the initiation {sigma} factor. The liberated RNA polymerase can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that removes undesirable nucleic acids from RNA polymerase.

molecular biology↗

bioRxiv2020

Markers of Acute Toxicity in Pancreatic Beta-Cells Exposed to Lethal Doses of the Organochlorine Pollutant DDT Determined by a Proteomic Approach

Many compounds have the potential to harm pancreatic beta-cells; organochlorine pollutants belong to those compounds. In this work, we aimed to find markers of acute toxicity of p,p -DDT among proteins expressed in human pancreatic beta-cells NES2Y and visible at 2-D electrophoresis. We exposed NES2Y cells to a lethal dose of p,p -DDT (150 M) for 24 hours and 30 hours and determined changes in protein expression using 2-D electrophoresis. We also stained the cells exposed to p,p -DDT to visualize the altered phenotype of the exposed cells. Among proteins with changed expression, we identified proteins involved in ER stress (GRP78, and endoplasmin), mitochondrial proteins (GRP75, ECHM, IDH3A, NDUS1, and NDUS3), proteins with potential to change the cell morphology (EFHD2, TCPA, NDRG1, and ezrin), and some other proteins (HNRPF, HNRH1, K2C8, vimentin, PBDC1, EF2, PCNA, biliverdin reductase, G3BP1, FRIL, and HSP27). These proteins can be used as markers of acute DDT toxicity.

pharmacology and toxicology↗