Biology subjects

Malinen, A. M.

Publications and source records attributed to Malinen, A. M..

7 recordsLinked to original sources

bioRxiv2026

A multimodal, correlative magnetic tweezers-TIRF platform for high-throughput single-molecule interrogations

Single-molecule techniques can resolve biological reactions at unmatched detail, but their low throughput and single-modality readouts have kept them out of data-intensive pipelines such as omics and drug discovery, and beyond reach of low-yield biological systems. Here we introduce a multimodal platform integrating high-throughput magnetic tweezers with ultra-wide-and flat-field objective-based total internal reflection fluorescence, enabling simultaneous force, torque, multicolor fluorescence, and temperature-dependent measurements on up to thousands of individual molecules in parallel and in real time. We demonstrate accurate single-molecule Forster resonance energy transfer (smFRET) for prism-based spectral imaging, capture temperature-dependent hairpin folding dynamics at high temporal resolution with smFRET and use correlative torque-fluorescence measurements to unravel the open-complex formation dynamics during bacterial transcription initiation. By unifying high resolution, throughput, and multimodal readout, this platform enables multidimensional dissection of complex biomolecular reactions with high statistical confidence, unlocking single-molecule biophysics for integration with drug discovery, omics, and cryo-EM workflows.

biophysics↗

bioRxiv2025

A kinetic ruler controls mRNA poly(A) tail length

Poly(A) tails of newly synthesized mRNAs have uniform lengths, arising through cooperation between the cleavage and polyadenylation complex (CPAC) and poly(A) binding proteins (PABPs). In the budding yeast, Saccharomyces cerevisiae, the responsible PABP is the evolutionarily conserved CCCH zinc finger protein Nab2 that facilitates the biogenesis of [~]60 adenosine mRNA poly(A) tails. Here, we address the molecular basis for such length control. Reconstituting polyadenylation reactions during the formation of Nab2:poly(A) RNA ribonucleoprotein particles in vitro, we find that Nab2 dimerization directs polyadenylation termination. The Nab2 dimer is stable only on poly(A) tails that are longer than 25 adenosines, explaining how Nab2 avoids prematurely terminating poly(A) synthesis. However, the mature tail length is not determined by the footprint of Nab2 on the RNA, but rather by the kinetic competition between CPAC-mediated tail elongation and Nab2 RNA-binding. Variations in Nab2 RNA-binding rate can shift poly(A) tail lengths, but in cells such variations are buffered by autoregulation of Nab2 protein concentration. As a result, poly(A) tail length control operates through a "kinetic ruler" mechanism, whereby the concentration of Nab2 quantifies RNA length.

molecular biology↗

bioRxiv2024

Determinants of transcription initiation efficiency and start site selection by RNA polymerase I

RNA polymerase I (Pol I) synthesizes pre-ribosomal RNA, which is essential for ribosome biogenesis. Increased Pol I activity supports rapid cell growth, a key characteristic of cancer cells. Consequently, drugs targeting Pol I in cancer are being actively investigated. The first step in pre-rRNA synthesis involves the assembly of the Pol I transcription initiation complex on the rRNA gene promoter, yet the quantitative and dynamic parameters of this process remain inadequately defined. We combined biochemical, biophysical, and molecular dynamics simulation analyses to enhance molecular models illustrating how Pol I apparatus isolated Saccharomyces cerevisiae recognizes the promoter and the transcription start site (TSS). Time-resolved binding data shows that CF relies on a two-step mechanism, consisting of the binding and isomerization steps, to recognize its binding site on the promoter. The next step, CF-mediated recruitment of the Pol I- Rrn3 complex to the promoter, was inefficient, leading to the rapid dissociation of Pol I. The selection of the TSS by Pol I is influenced by the distance to CF and the properties of downstream DNA, such as bendability. The dynamics revealed in the Pol I transcription apparatus establish a framework for comparing the functions and mechanisms of Pol I activators and inhibitors.

biochemistry↗

bioRxiv2021

Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor{sigma} to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein-DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RPO is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting with{sigma} and the RNAP {beta} subunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates.

biophysics↗

bioRxiv2021

The formation of the bacterial RNA polymerase-promoter open complex involves a branched pathway

The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)-{sigma}70 holoenzyme to the promoter DNA. This initial RNAP-promoter closed complex undergoes a series of conformational changes, including the formation of a transcription bubble on the promoter and the loading of template DNA strand into the RNAP active site; these changes lead to the catalytically active open complex (RPO) state. Recent cryo-electron microscopy studies have provided detailed structural insight on the RPO and putative intermediates on its formation pathway. Here, we employ single-molecule fluorescence microscopy to interrogate the conformational dynamics and reaction kinetics during real-time RPO formation. We find that the RPO pathway is branched, generating RPO complexes with different stabilities. The RNAP cleft loops, and especially the {beta} rudder, stabilise the transcription bubble. The RNAP interactions with the promoter upstream sequence (beyond -35) stimulate transcription bubble nucleation and tune the reaction path towards stable forms of the RPO. The mechanistic heterogeneity of the RPO pathway may be a prerequisite for its regulation since such heterogeneity allows the amplification of small promoter sequence or transcription-factor-dependent changes in the free energy profile of the RPO pathway to large differences in transcription efficiency.

biochemistry↗

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

Pausing controls branching between productive and non-productive pathways during initial transcription

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis by the bacterial RNA polymerase (RNAP) has been shown to be interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Here, we employed single-molecule FRET and biochemical analysis to disentangle the pausing-related pathways of bacterial initial transcription. We present further evidence that region {sigma}3.2 constitutes a barrier after the initial transcribing complex synthesizes a 6-nt RNA (ITC6), halting transcription. We also show that the paused ITC6 state acts as a checkpoint that directs RNAP, in an NTP-dependent manner, to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway that blocks transcription initiation. Our results show that abortive RNA release and DNA unscrunching are not as tightly coupled as previously thought.

biophysics↗