Biology subjects

Qayyum, M. Z.

Publications and source records attributed to Qayyum, M. Z..

7 recordsLinked to original sources

bioRxiv2026

Shaping cryogenic 3D volume imaging by pFIB ion species and milling voltage

Cryogenic plasma focused ion beam-scanning electron microscopy (cryo-pFIB-SEM) enables nanoscale volume imaging of vitrified cells and tissues, but the fidelity of each newly exposed block-face depends on how ions interact with compositionally heterogeneous biological material. Using xenon, argon, oxygen, and nitrogen plasma beams across a 30-2 kV voltage range, we examined how ion species and accelerating voltage influence block-face quality and ultrastructural fidelity. Curtaining imposed a species-dependent lower limit on accelerating voltage, with xenon and argon supporting uniform milling to 10 kV and oxygen and nitrogen to 15 kV. Monte Carlo simulations predicted substantially reduced ion penetration and atomic displacement under these species-specific low-kV conditions. Experimentally, lowering milling voltage suppressed charging and topographic artifacts, with the largest effects observed at heterogeneous interfaces. Oxygen and nitrogen produced greater membrane sharpness and organelle contrast than the noble gases, and these differences influenced machine-learning-based recognition of fine organelle features in 3D datasets. Together, these findings show that cryo-pFIB-SEM image quality cannot be optimized by accelerating voltage or ion species independently. Instead, milling performance reflects a tradeoff among surface fidelity, charging, contrast, and curtaining that depends on local specimen composition. Species-specific low-kV milling therefore provides a practical framework for matching cryo-pFIB-SEM acquisition conditions to the structural and biophysical properties of the biological target.

cell biology↗

bioRxiv2026

Depth-resolved analysis reveals target-dependent cryo-FIB damage profiles

Cryogenic focused ion beam (cryo-FIB) milling enables cellular cryo-electron tomography but can compromise high-resolution structural information near milled surfaces. Here we establish recombinant human apoferritin (apoF) as a reporter of depth-dependent structural preservation in cellular lamellae. ApoF yielded a 2.05 [A] in situ reconstruction. Apparent FIB damage was most pronounced within the first 15 nm for xenon, 30 nm for gallium, 45 nm for argon and remained detectable at least 75 nm from oxygen-milled surfaces under the workflows tested. Endogenous 70S ribosomes in the same xenon-milled tomograms showed a broader 30-45 nm profile, whereas {beta}-galactosidase showed a similar narrow xenon-associated profile in a different specimen. Xenon-milled lamellae 125-150 nm thick yielded the highest resolution among the thickness groups examined, with lower resolution in the thinner and thickest groups. These findings show structural preservation is constrained by both surface-associated damage and lamella thickness, with the apparent extent of damage varying by molecular reporter. Previous ribosome-based surface-exclusion estimates may therefore be overly conservative for some molecular targets.

molecular biology↗

bioRxiv2024

Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, {beta}1 and {beta}2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spans approximately half of the {beta}2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding at the active site results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms. Significance statementCellular RNA polymerase (RNAP) carries out RNA synthesis and proofreading reactions utilizing a mobile catalytic domain known as the trigger loop/helix. In cyanobacteria, this essential domain acquired a large Si3 insertion during the course of evolution. Despite its elongated shape and large swinging motion associated with the transition between the trigger loop and helix, Si3 is effectively accommodated within cyRNAP, with no impact on the fundamental functions of the trigger loop. Understanding the significance of Si3 in cyanobacteria and chloroplasts is expected to reveal unique transcription mechanism in photosynthetic organisms.

biochemistry↗

bioRxiv2022

Allosteric mechanism of transcription inhibition by NusG-dependent pausing of RNA polymerase

NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteriaincluding Bacillus subtilis by sequence-specific interaction with a conserved pause-inducing -11TTNTTT-6 motif found in the non-template DNA (ntDNA) strand within the transcription bubble. To reveal the structural basis of NusG-dependent pausing, we determined a cryo-EM structure of a paused transcription complex containing RNAP, NusG, and the TTNTTT motif in the ntDNA strand. Interaction of NusG with the ntDNA strand rearranges the transcription bubble by positioning three consecutive T residues in a cleft between NusG and the {beta}-lobe domain of RNAP. We revealed that the RNAP swivel module rotation (swiveling), which widens (swiveled state) and narrows (non-swiveled state) a cleft between NusG and the {beta}-lobe, is an intrinsic motion of RNAP and is directly linked to nucleotide binding at the active site and to trigger loop folding, an essential conformational change of all cellular RNAPs for the RNA synthesis reaction. We also determined cryo-EM structures of RNAP escaping from a paused transcription complex. These structures revealed the NusG-dependent pausing mechanism by which NusG-ntDNA interaction inhibits the transition from swiveled to non-swiveled states, thereby preventing trigger loop folding and RNA synthesis allosterically. This motion is also reduced by formation of an RNA hairpin within the RNA exit channel. Thus, the pause half-life can be modulated by the strength of the NusG-ntDNA interaction and/or the stability of the RNA hairpin. NusG residues that interact with the TTNTTT motif are widely conserved in bacteria, suggesting that NusG-dependent pausing of transcription is widespread. Significance statementTranscription pausing by RNA polymerase (RNAP) regulates gene expression where it controls co-transcriptional RNA folding, synchronizes transcription with translation, and provides time for binding of regulatory factors. Transcription elongation factor NusG stimulates pausing in Gram+ bacteriaincluding Bacillus subtilis and Mycobacterium tuberculosis by sequence-specific interaction with a conserved pause motif found in the non-template DNA (ntDNA) strand within the transcription bubble. Our structural and biochemical results revealed that part of the conserved TTNTTT motif in ntDNA is extruded and sandwiched between NusG and RNAP. Our results further demonstrate that an essential global conformational change in RNAP is directly linked to RNA synthesis, and that the NusG-ntDNA interaction pauses RNA synthesis by interfering with this conformational change.

molecular biology↗

bioRxiv2021

Structural basis of RNA polymerase recycling by the Swi2/Snf2 ATPase RapA in Escherichia coli

After transcription termination, cellular RNA polymerases (RNAPs) are occasionally trapped on DNA, impounded in an undefined Post-Termination Complex (PTC), limiting free RNAP pool and making transcription inefficient. In Escherichia coli, a Swi2/Snf2 ATPase RapA is involved in countering such inefficiency through RNAP recycling. To understand its mechanism of RNAP recycling, we have determined the cryo-electron microscopy (cryo-EM) structures of two sets of E. coli RapA-RNAP complexes along with RNAP core enzyme and elongation complex (EC). The structures revealed the large conformational changes of RNAP and RapA upon their association implicated in the hindrance in PTC formation. Our study reveals that although RapA binds away from the DNA binding channel of RNAP, it can close the RNAP clamp allosterically thereby preventing its non-specific DNA binding. Together with DNA binding assays, we propose that RapA acts as a guardian of RNAP by which prevents non-specific DNA binding of RNAP without affecting the sigma factor binding to RNAP core enzyme, thereby enhancing RNAP recycling.

biochemistry↗

bioRxiv2020

Structural basis of ribosomal RNA transcription regulation

Ribosomal RNA (rRNA) is the most highly expressed gene in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. To reveal the mechanism of highly regulated rRNA transcription, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) {sigma}70 holoenzyme at different steps of rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element of rRNA promoter using the dimerized subunit carboxyl-terminal domain and scrunches the template DNA with the {sigma}finger and {beta}lid to select a transcription start site favorable for rRNA expression. Promoter DNA binding to RNAP induces conformational change of the {sigma} domain 2 that opens a gate for DNA loading and ejects {sigma}1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding to RNAP also opens the DNA loading gate, but it is not coupled to {sigma}1.1 ejection and impedes the open complex formation of the rRNA promoter due to its G+C rich discriminator sequence. Mutations in {sigma}1.1 or the {beta}lid stabilize the RNAP and rRNA promoter complex and decrease its sensitivity to DksA/ppGpp. These results provide a molecular basis for exceptionally active rRNA transcription and for its vulnerability to DksA/ppGpp.

molecular biology↗

bioRxiv2018

Cryo-EM structure of Escherichia coli σ70 RNAP and promoter DNA complex revealed a role of σ nonconserved region during the open complex formation

First step of gene expression is transcribing the genetic information stored in DNA to RNA by the transcription machinery including RNA polymerase (RNAP). In Escherichia coli, a primary {sigma}70 factor form the RNAP holoenzyme to express housekeeping genes. The {sigma}70 contains a large insertion at between the conserved regions 1.2 and 2.1, the {sigma} non-conserved region ({sigma}NCR), but its function remains to be elucidated. In this study, we determined the cryo-EM structures of the E. coli RNAP {sigma}70 holoenzyme and its complex with promoter DNA (open complex, RPo) at 4.2 and 5.75 [A] resolutions, respectively, to reveal native conformations of RNAP and DNA. The RPo structure presented here found an interaction between R157 residue in the {sigma}NCR and promoter DNA just upstream of the -10 element, which was not observed in a previously determined E. coli RNAP transcription initiation complex (RPo plus short RNA) structure by X-ray crystallography due to restraint of crystal packing effect. Disruption of the {sigma}NCR and DNA interaction by the amino acid substitution (R157E) influences the DNA opening around the transcription start site and therefore decreases the transcription activity of RNAP. We propose that the {sigma}NCR and DNA interaction is conserved in proteobacteria and RNAP in other bacteria replace its role with a transcription factor.

molecular biology↗