Characterization and DNA Stable-Isotope Probing of Methanotrophic Bioaerosols.

The growth and activity of bacteria have been extensively studied in nearly every environment on Earth, but there have been limited studies focusing on the air. Suspended bacteria (outside of water droplets) may stay in the atmosphere for time frames that could allow for growth on volatile compounds, including the potent greenhouse gas methane. We investigated the ability of aerosolized methanotrophic bacteria to grow on methane in the airborne state in rotating gas-phase bioreactors.

Year-long wastewater monitoring for SARS-CoV-2 signals in combined and separate sanitary sewers.

COVID-19 wastewater-based epidemiology has been performed in catchments of various sizes and sewer types with many short-term studies available and multi-seasonal studies emerging. The objective of this study was to compare weekly observations of SARS-CoV-2 genes in municipal wastewater across multiple seasons for different systems as a factor of sewer type (combined, separate sanitary) and system size. Sampling occurred following the first wave of SARS-CoV-2 cases in the study region (June 2020) and continued through the third wave (May 2021), the period during which clinical testing was widely available and different variants dominated clinical cases.

Drug Repositioning: A Unique Approach to Refurbish Drug Discovery.

For a decade, it has been observed that there is a remarkable decrease in the quantum of novel clinically approved drugs, in spite of modernization in the research and development process. We have highlighted repositioning of drugs as a methodology that has found new therapeutic implications for clinically approved drugs but with different indications. This can be considered as an upbringing strategy to deliver timely and cost-effective solutions, which still need exploration for getting over the shortage of novel drugs reaching the market.

The dynamic landscape of transcription initiation in yeast mitochondria.

Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution.

The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation.

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides.

Mechanisms of template handling and pseudoknot folding in human telomerase and their manipulation to expand the sequence repertoire of processive repeat synthesis.

Telomerase adds telomeric repeats to chromosome ends by processive copying of a template within the telomerase RNA bound to telomerase reverse transcriptase. Telomerase RNAs have single-stranded regions that separate the template from a 5' stem and 3' pseudoknot, and mammals gained additional stem P2a.1 separating the template from the pseudoknot.

The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA.

Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1.

Interactions of the yeast mitochondrial RNA polymerase with the +1 and +2 promoter bases dictate transcription initiation efficiency.

Mitochondrial promoters of Saccharomyces cerevisiae share a conserved -8 to +1 sequence with +1+2 AA, AG or AT initiation sequence, which dictates the efficiency of transcription initiation by the mitochondrial RNA polymerase Rpo41 and its initiation factor Mtf1. We used 2-aminopurine fluorescence to monitor promoter melting and measured the kcat/Km of 2-mer synthesis to quantify initiation efficiency with systematic changes of the +1+2 base pairs to matched and mismatched pairs. We show that AA promoters are most efficient, followed by AG and then AT promoters, and the differences in their efficiencies stem specifically from differential melting of +1+2 region without affecting melting of the upstream -4 to -1 region.

Fluorescent methods to study transcription initiation and transition into elongation.

The DNA-dependent RNA polymerases induce specific conformational changes in the promoter DNA during transcription initiation. Fluorescence spectroscopy sensitively monitors these DNA conformational changes in real time and at equilibrium providing powerful ways to estimate interactions in transcriptional complexes and to assess how transcription is regulated by the promoter DNA sequence, transcription factors, and small ligands. Ensemble fluorescence methods described here probe the individual steps of promoter binding, bending, opening, and transition into the elongation using T7 phage and mitochondrial transcriptional systems as examples.

Mechanism of transcription initiation by the yeast mitochondrial RNA polymerase.

Mitochondria are the major supplier of cellular energy in the form of ATP. Defects in normal ATP production due to dysfunctions in mitochondrial gene expression are responsible for many mitochondrial and aging related disorders. Mitochondria carry their own DNA genome which is transcribed by relatively simple transcriptional machinery consisting of the mitochondrial RNAP (mtRNAP) and one or more transcription factors.

Transcription factor-dependent DNA bending governs promoter recognition by the mitochondrial RNA polymerase.

Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1).

The N-terminal domain of the yeast mitochondrial RNA polymerase regulates multiple steps of transcription.

Transcription of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) genome is catalyzed by nuclear-encoded proteins that include the core RNA polymerase (RNAP) subunit Rpo41 and the transcription factor Mtf1. Rpo41 is homologous to the single-subunit bacteriophage T7/T3 RNAP. Its ∼80-kDa C-terminal domain is highly conserved among mt RNAPs, but its ∼50-kDa N-terminal domain (NTD) is less conserved and not present in T7/T3 RNAP.