Biology subjects

Mazumder, A.

Publications and source records attributed to Mazumder, A..

30 records · Page 2Linked to original sources

bioRxiv2021

Transcription initiation at a consensus bacterial promoter proceeds via a "bind-unwind-load-and-lock" mechanism

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, "bind-unwind-load-and-lock," model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters. Significance statementTranscription initiation, the first step and most important step in gene expression for all organisms, involves unwinding of promoter DNA by RNA polymerase (RNAP) to form an open complex (RPo); this step also underpins transcriptional regulation and serves as an antibiotic target. Despite decades of research, the mechanism of promoter DNA unwinding has remained unresolved. Here, we solve this puzzle by using single-molecule fluorescence to directly monitor conformational changes in the promoter DNA and RNAP in real time during RPo formation. We show that RPo forms via a "bind-unwind-load-and-lock" mechanism, where the promoter unwinds outside the RNAP cleft, the unwound template DNA loads into the cleft, and RNAP "locks" the template DNA in place by closing the RNAP clamp module.

biochemistry↗

bioRxiv2020

Race specific differences in DNA damage repair dysregulation in breast cancer and association with outcome

IMPORTANCEAfrican American (AA) breast cancer patients have worse outcomes than Caucasian Americans (CAs). DNA damage repair (DDR) genes drive poor outcome in CA estrogen receptor (ER)+ breast cancer patients. Whether DDR genes similarly impact survival in AAs is unknown. Identifying AA-specific patterns of DDR dysregulation could change how we tailor predictive/prognostic biomarkers. OBJECTIVETo characterize DDR dysregulation in ER+ AA patient tumors and test associations with clinical outcome. DESIGN SETTINGS AND PARTICIPANTSThree independent tumor, and two normal breast datasets were analyzed. Tumor datasets: (1) GSE78958 (2) GSE18229 (3) The Cancer Genome Atlas (TCGA). Normal datasets: (4) GSE43973 (5) GSE50939. MAIN OUTCOME AND MEASURESUp/down-regulation of 104 DDR genes was assessed in AA samples vs CAs. Survival associations were assessed for genes dysregulated in multiple datasets. RESULTSOverall, RNA levels of single strand break repair (SSBR) genes were downregulated in AA tumors and double strand break repair (DSBR) genes were upregulated compared to CAs. While SSBR downregulation was mainly detected in tumors, DSBR upregulation was detectable in both tumor and normal breast AA samples. Seven specific DDR genes identified as dysregulated in AAs vs CAs in multiple datasets associated with poor survival. A subset of tumors with simultaneous dysregulation of homologous recombination and single strand break repair genes was enriched in AAs and had associated consistently with poor survival. CONCLUSION AND RELEVANCEOverall, these results constitute the first systematic analysis of differences in DDR regulation in AA ER+ tumors and normal tissue vs CAs. We identify a profile of DDR dysregulation enriched in AA patients, which associates with poor outcome. These results suggest a distinct molecular mechanism of DDR regulation in AAs that lays the groundwork for refining biomarker profiles by race and improving precision medicine for underserved populations.

cancer biology↗

bioRxiv2020

RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding

The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Forster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes [~] 0.3-0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes >1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.

biophysics↗

bioRxiv2020

Comparative analysis of chloroplast genomes indicated different origin for Indian Tea (Camellia assamica) cv TV-1 as compared to Chinese tea

Tea is an important plantation crop of some Asian and African countries. Based upon the morphological characteristics, tea is classified botanically into 2 main types i.e. Assam and China, which are morphologically very distinct. Further, they are so easily pollinated among themselves, that a third category, Cambod type is also described. Although the general consensus of origin of tea is India, Burma and China joining area, yet specific origin of China and Assam tea are not yet clear. In the present study, we made an attempt to understand the origin of Indian tea through the comparative analysis of different chloroplast (cp) genomes under the Camellia genus. Cp genome based phylogenetic analysis indicated that Indian Assam Tea, TV-1 formed a different group from that of China tea, indicating that TV-1 might have undergone different domestication and hence owe different origin. The simple sequence repeats (SSRs) analysis and codon usage distribution pattern also supported the clustering order in the cp genome based phylogenetic tree.

genomics↗

bioRxiv2020

Mismatch repair deficiency predicts response to HER2 blockade in HER2-negative breast cancer

Estrogen receptor positive (ER+) breast cancer is a leading cause of cancer-related death globally. Resistance to standard of care endocrine treatment occurs in at least 30% of ER+ breast cancer patients resulting in ~40,000 deaths every year in the US alone. Preclinical studies strongly implicate activation of growth factor receptor, HER2 in endocrine treatment resistance of ER+ breast cancer that is HER2- at diagnosis1,2. However, clinical trials of pan-HER inhibitors in ER+/HER2- patients have disappointed, likely due to a lack of predictive biomarkers3-6. Here we demonstrate that loss of MLH1, a principal mismatch repair gene, causally activates HER2 in ER+/HER2- breast cancer upon endocrine treatment. Additionally, we show that HER2 activation is indispensable for endocrine treatment resistant growth of MLH1- cells in vitro and in vivo. Consequently, inhibiting HER2 restores sensitivity to endocrine treatment in multiple experimental models including patient-derived xenograft tumors. Patient data from multiple clinical datasets (TCGA, METABRIC, Alliance (Z1031) and E-GEOD-28826) supports an association between MLH1 loss, HER2 upregulation, and sensitivity to trastuzumab in endocrine treatment-resistant ER+/HER2- patients. These results provide strong rationale that MLH1 could serve as a first-in-class predictive marker of sensitivity to combinatorial treatment with endocrine drugs and HER inhibitors in endocrine treatment-resistant ER+/HER2- breast cancer patients. Implications of this study extend beyond breast cancer to Lynch Syndrome cancers. One Sentence SummaryDefective mismatch repair activates HER2 in HER2-negative breast cancer cells and renders them susceptible to HER2 inhibitors.

cancer biology↗

bioRxiv2020

Biocomputational Analysis Establishes Genetic Association of Rheumatoid Arthritis (RA) and Migraine

An autoimmune disease and a neurological disease do not tie up together but if statistics say that 61% of people are affected by Rheumatoid Arthritis (an autoimmune disease) and Migraine (a neurological disease) at the same time it is really a point to be noted. The previous studies show that Rheumatoid Arthritis (RA) and Migraine are related anatomically but our goal was to dig out any other similarities except it. Therefore, we studied their genes and hoped to get something fascinating. We assessed the genes from the literature study and Genetic Home Reference (https://ghr.nlm.nih.gov/). After that, the genetic sequences of these genes were extracted from NCBI (National Center for Biotechnology Information, http://ncbi.nlm.nih.gov) and by applying multiple sequence alignment, we created a phylogenetic tree by Mega (Molecular Evolutionary Genetics Analysis). Surprisingly, we found 10 pairs of genes with similar genetic structures and common ancestry. Therefore, we can say these 10 pairs of genes are related to each other closely, which is why Migraine and Rheumatoid Arthritis (RA) are found together in a person. At the last part of this study, we used protein expression for the evaluation of the result. Here our bioinformatics approach may help to strengthen the connectedness of these two diverse diseases.

bioinformatics↗

bioRxiv2019

Closing and opening of the RNA polymerase trigger loop

The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an \"unfolded\"/\"open\" state that allows an NTP substrate to enter the active center and a \"folded\"/\"closed\" state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription elongation.

molecular biology↗

bioRxiv2019

The γH2AX Peak in the S Phase After UV Irradiation Corresponds to the sites of DNA Replication and not DNA Damage

Ultraviolet (UV) radiation is a major environmental mutagen. Exposure to UV leads to a sharp peak of γH2AX – the phosphorylated form of a histone variant H2AX – in the S phase within an asynchronous population of cells. γH2AX is often considered as a definitive marker of DNA damage inside a cell. In this report we show that γH2AX in the S phase cells after UV irradiation does not report on the extent of primary DNA damage in the form of cyclobutane pyrimidine dimers or on the extent of its secondary manifestations as DNA double strand breaks or in the inhibition of global transcription. Instead γH2AX in the S phase corresponds to the sites of active replication at the time of UV irradiation – despite which, the cells complete the replication of their genomes and arrest within the G2 phase. Moreover, cells in all the phases of the cell cycle develop similar levels of DNA damage. Our study suggests that it is not DNA damage but the response elicited, which peaks in the S phase upon UV damage.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2019

Investigating the Central Dogma of Molecular Biology in the Context of Nuclear Architecture and Cell Cycle

Nuclear architecture is the organization of the genome within a cell nucleus with respect to different nuclear landmarks such as nuclear lamina, matrix or nucleoli. Lately it has emerged as a major regulator of gene expression in mammalian cells. The studies connecting nuclear architecture with gene expression are largely population-averaged and do not report on the heterogeneity in genome organization or in gene expression within a population. In this report we present a method for combining 3D DNA Fluorescence in situ Hybridization (FISH) with single molecule RNA FISH (smFISH) and immunofluorescence to study nuclear architecture-dependent gene regulation on a cell-by-cell basis. We further combine it with an imaging-based cell cycle staging to correlate nuclear architecture with gene expression across the cell cycle. We present this in the context of Cyclin A2 (CCNA2) gene for its known cell cycle-dependent expression. We show that, across the cell cycle, the expression of a CCNA2 gene copy is stochastic and depends neither on its sub-nuclear position--which usually lies close to nuclear lamina--nor on the expression from the other copies.

cell biology↗

bioRxiv2018

One-dimensional sliding assists σ70-dependent promoter binding by Escherichia coli RNA polymerase

The search for a promoter on DNA by RNA polymerase (RNAP) is an obligatory first step in transcription. The role of facilitated diffusion during promoter search has been controversial. Here, we re-assessed facilitated diffusion in promoter search by imaging motions of single molecules of Escherichia coli RNAP {sigma}70 holoenzyme on single DNA molecules suspended between optical traps in a manner that absolutely avoided interactions with surfaces. The assay enabled us to observe unambiguous one-dimensional sliding of RNAP {sigma}70 holoenzyme for thousands of DNA base pairs during promoter search. Analysis of binding kinetics revealed short binding events on nonspecific DNA (0.4 s), intermediate binding events on A/T-rich DNA (1.6 s), and long binding events at or near promoters (>300 s). We estimate a lower bound for the \"diffusion facilitation threshold\" - the RNAP concentration at which three-dimensional search and one-dimensional sliding contribute equally to promoter binding - of 0.2 M RNAP. The results suggest facilitated diffusion occurs in promoter search by RNAP, even at the relatively high, 0.2-0.6 M, concentrations of RNAP in cells.\n\nSignificance statementThe flow of genetic information from DNA to RNA is of central importance to living systems, and it can only start after an RNA polymerase (RNAP) has found a promoter site. But how does this enzyme find promoter sites on DNA in the first place? In recent years, debate on this topic has favored a promoter search mechanism that is dominated by three-dimensional diffusion of RNAP, rather than by one-dimensional sliding of RNAP on DNA. Here, we designed an improved single-molecule assay that unambiguously revealed extensive one-dimensional sliding of RNAP on DNA. Our results imply that, at the RNAP concentrations in living cells, the promoter-search process is facilitated by one-dimensional sliding on DNA.

biophysics↗

bioRxiv2018

The RNA polymerase clamp interconverts dynamically among three states and is stabilized in a partly closed state by ppGpp

RNA polymerase (RNAP) contains a mobile structural module, the \"clamp,\" that forms one wall of the RNAP active-center cleft and that has been linked to crucial aspects of the transcription cycle, including loading of promoter DNA into the RNAP active-center cleft, unwinding of promoter DNA, transcription elongation complex stability, transcription pausing, and transcription termination. Crystal structures and single-molecule FRET studies establish that the clamp can adopt open and closed conformational states; however, the occurrence, pathway, and kinetics of transitions between clamp states have been unclear. Using single-molecule FRET (smFRET) on surface-immobilized RNAP molecules, we show that the clamp in RNAP holoenzyme exists in three distinct conformational states: the previously defined open state, the previously defined closed state, and a previously undefined partly closed state. smFRET time-traces show dynamic transitions between open, partly closed, and closed states on the 0.1-1 second time-scale. Similar analyses of transcription initiation complexes confirm that the RNAP clamp is closed in the catalytically competent transcription initiation complex and in initial transcribing complexes (RPITC), including paused initial transcribing complexes, and show that, in these complexes, in contrast to in RNAP holoenzyme, the clamp does not interconvert between the closed state and other states. The stringent-response alarmone ppGpp selectively stabilizes the partly-closed-clamp state, inhibiting interconversion between the partly closed state and the open state. The methods of this report should allow elucidation of clamp conformation and dynamics during all phases of transcription.\n\nSIGNIFICANCE STATEMENTThe clamp forms a pincer of the RNA polymerase \"crab-claw\" structure, and adopts many conformations with poorly understood function and dynamics. By measuring distances within single surface-attached molecules, we observe directly the motions of the clamp and show that it adopts an open, a closed, and a partly closed state; the last state is stabilized by a sensor of bacterial starvation, linking the clamp conformation to the mechanisms used by bacteria to counteract stress. We also show that the clamp remains closed in many transcription steps, as well as in the presence of a specific antibiotic. Our approach can monitor clamp motions throughout transcription and offers insight on how antibiotics can stop pathogens by blocking their RNA polymerase movements.

biophysics↗

bioRxiv2017

Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

Fidaxomicin is an antibacterial drug in clinical use in treatment of Clostridium difficile diarrhea1-2. The active pharmaceutical ingredient of fidaxomicin, lipiarmycin A3 (Lpm)1-4, is a macrocyclic antibiotic with bactericidal activity against Gram-positive bacteria and efflux-deficient strains of Gram-negative bacteria1-2, 5. Lpm functions by inhibiting bacterial RNA polymerase (RNAP)6-8. Lpm exhibits no cross-resistance with the classic RNAP inhibitor rifampin (Rif)7, 9 and inhibits transcription initiation at an earlier step than Rif8-11, suggesting that the binding site and mechanism of Lpm differ from those of Rif. Efforts spanning a decade to obtain a crystal structure of RNAP in complex with Lpm have been unsuccessful. Here, we report a cryo-EM12-13 structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5 [A] resolution. The structure shows that Lpm binds at the base of the RNAP \"clamp,\" interacting with the RNAP switch region and the RNAP RNA exit channel. The binding site on RNAP for Lpm does not overlap the binding sites for other RNAP inhibitors, accounting for the absence of cross-resistance of Lpm with other RNAP inhibitors. The structure exhibits an open conformation of the RNAP clamp, with the RNAP clamp swung outward by ~17{degrees} relative to its position in catalytically competent RNAP-promoter transcription initiation complexes, suggesting that Lpm traps an open-clamp conformational state. Single-molecule fluorescence resonance energy transfer14 experiments confirm that Lpm traps an open-clamp conformational state and define effects of Lpm on clamp opening and closing dynamics. We propose that Lpm inhibits transcription initiation by trapping an open-clamp conformational state, thereby preventing simultaneous engagement of transcription initiation factor {sigma} regions 2 and 4 with promoter -10 and -35 elements. The results provide information essential to understanding the mode of action of Lpm, account for structure-activity relationships of known Lpm analogs, and suggest modifications to Lpm that could yield new, improved Lpm analogs.

molecular biology↗