The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility

The EMBO journal EMBO Press 40:6 (2021) e104683

Authors:

Oshri Afanzar, Diana Di Paolo, Miriam Eisenstein, Kohava Levi, Anne Plochowietz, Achillefs N Kapanidis, Richard Michael Berry, Michael Eisenbach

Abstract:

Regulatory switches are wide spread in many biological systems. Uniquely among them, the switch of the bacterial flagellar motor is not an on/off switch but rather controls the motor's direction of rotation in response to binding of the signaling protein CheY. Despite its extensive study, the molecular mechanism underlying this switch has remained largely unclear. Here, we resolved the functions of each of the three CheY-binding sites at the switch in E. coli, as well as their different dependencies on phosphorylation and acetylation of CheY. Based on this, we propose that CheY motor switching activity is potentiated upon binding to the first site. Binding of potentiated CheY to the second site produces unstable switching and at the same time enables CheY binding to the third site, an event that stabilizes the switched state. Thereby, this mechanism exemplifies a unique combination of tight motor regulation with inherent switching flexibility.

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

Nucleic Acids Research Oxford University Press 49:5 (2021) 2790-2802

Authors:

Abhishek Mazumder, Anna Wang, Heesoo Uhm, Richard H Ebright, Achillefs N Kapanidis

Abstract:

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 Förster 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.

Amplification-Free Detection of Viruses in Minutes using Single-Particle Imaging and Machine Learning

Biophysical Journal Elsevier 120:3 (2021) 195a

Authors:

Nicolas Shiaelis, Leon Peto, Andrew McMahon, Chritof Hepp, Erica Bickerton, Cyril Favard, Delphine Muriaux, Monique Andersson, Alison Vaughan, Philippa Matthews, Nicole Stoesser, Derrick Crook, Achillefs N Kapanidis, Nicole C Robb

High-Throughput Super-Resolution Microscopy of Viral Particles Reveals Insights into their Morphology and Organisation

(2021)

Authors:

Andrew McMahon, Christof Hepp, Nicole C Robb

Quantification of purified endogenous miRNAs with high sensitivity and specificity

Nature Communications Springer Nature 11:1 (2020) 6033

Authors:

Soochul Shin, Yoonseok Jung, Heesoo Uhm, Minseok Song, Soomin Son, Jiyoung Goo, Cherlhyun Jeong, Ji-Joon Song, V Narry Kim, Sungchul Hohng

Abstract:

MicroRNAs (miRNAs) are short (19–24 nt) non-coding RNAs that suppress the expression of protein coding genes at the post-transcriptional level. Differential expression profiles of miRNAs across a range of diseases have emerged as powerful biomarkers, making a reliable yet rapid profiling technique for miRNAs potentially essential in clinics. Here, we report an amplification-free multi-color single-molecule imaging technique that can profile purified endogenous miRNAs with high sensitivity, specificity, and reliability. Compared to previously reported techniques, our technique can discriminate single base mismatches and single-nucleotide 3′-tailing with low false positive rates regardless of their positions on miRNA. By preloading probes in Thermus thermophilus Argonaute (TtAgo), miRNAs detection speed is accelerated by more than 20 times. Finally, by utilizing the well-conserved linearity between single-molecule spot numbers and the target miRNA concentrations, the absolute average copy numbers of endogenous miRNA species in a single cell can be estimated. Thus our technique, Ago-FISH (Argonaute-based Fluorescence In Situ Hybridization), provides a reliable way to accurately profile various endogenous miRNAs on a single miRNA sensing chip.