Biology subjects

Kipper, K.

Publications and source records attributed to Kipper, K..

4 recordsLinked to original sources

bioRxiv2022

Understanding emergence of antimycobacterial dose dependent resistance.

Concentration dependency of phenotypic and genotypic isoniazid-rifampicin resistance emergence was investigated to obtain a mechanistic understanding on how anti-mycobacterial drugs facilitate the emergence of bacterial populations that survive throughout treatment. Using static kill curve experiments, observing two evolution cycles, it was demonstrated that rifampicin resistance was the result of non-specific mechanisms and not associated with accumulation of drug resistance encoding SNPs. Whereas, part of isoniazid resistance could be accounted for by accumulation of specific SNPs, which was concentration dependent. Using a Hollow Fibre Infection Model it was demonstrated that emergence of genotypic resistance only occurs when antibiotic levels fall below MIC although MICs are typically maintained following clinical dosing provided that adherence to the regimen is good. This study showed that disentangling and quantifying concentration dependent emergence of resistance provides improved rational for drug and dose selection although further work on understanding underlying mechanisms is needed to improve the drug development pipeline. One Sentence SummaryDisentangling and quantifying concentration dependent emergence of resistance will contribute to better informed drug and dose selection for anti-mycobacterial combination therapy.

pharmacology and toxicology↗

bioRxiv2022

Neuronal RNA granules are ribosome complexes stalled at the pre-translocation state

The polarized cell morphology of neurons dictates many neuronal processes, including the axodendridic transport of specific mRNAs and subsequent translation. mRNAs together with ribosomes and RNA-binding proteins form RNA granules that are targeted to axodendrites for localized translation in neurons. It has been established that localized protein synthesis in neurons is essential for long-term memory formation, synaptic plasticity, and neurodegeneration. We have used proteomics and electron microscopy to characterize neuronal RNA granules (nRNAg) isolated from rat brain tissues or human neuroblastoma. We show that ribosome containing RNA granules are morula-like structures when visualized by electron microscopy. Crosslinking-coupled mass-spectrometry identified potential G3BP2 binding site on the ribosome near the eIF3d-binding site on the 40S ribosomal subunit. We used cryo-EM to resolve the structure of the ribosome-component of nRNAg. The cryo-EM reveals that ribosomes in the nRNAg are stalled at the elongation state where tRNAs are in the hybrid A/P and P/E site, and resemble the pre-translocation state ribosomes. We also describe a new kind of principal motion of the ribosome, which we call the rocking motion.

molecular biology↗

bioRxiv2020

Mechanism of DNA surface exploration and operator bypassing during target search

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific DNA1-5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore DNA during the 1D phase of target search. To track the rotation of sliding LacI molecules on the microsecond time scale during DNA surface search, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluorescence signal fluctuations are accurately described by rotation-coupled sliding, where LacI traverses ~40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA, suggesting that the sliding protein frequently hops out of the DNA groove, which would result in frequent bypassing of target sequences. Indeed, we directly observe such bypassing by single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI at most hops one to two grooves (10-20 bp) every 250 s. Overall, our data suggest a speed-accuracy trade-off during sliding; the weak nature of non-specific protein-DNA interactions underlies operator bypassing but also facilitates rapid sliding. We anticipate that our SMCT-FCS method to monitor rotational diffusion on the microsecond time scale while tracking individual molecules with millisecond time resolution will be applicable to the real-time investigation of many other biological interactions and effectively extends the accessible time regime by two orders of magnitude.

biophysics↗

bioRxiv2018

Direct observation of rotation-coupled protein diffusion along DNA on the microsecond timescale

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific regions of the DNA1-5. It is however not known how sliding proteins are oriented with respect to DNA in order to recognize specific sequences. Here we measure the polarization of fluorescence emission from single fluorescently labeled lac repressor (LacI) molecules sliding on stretched DNA. Real-time feedback-coupled confocal single-particle tracking allows us to measure fluorescence correlation of the sliding molecules. We find that the fluctuations in the fluorescence signal on the s timescale are accurately described by rotation-coupled sliding on DNA. On average, LacI moves [~]50 base pairs per revolution, which is significantly longer than the 10.5 bp helical periodicity of DNA. Our data support a facilitated diffusion model1 where the transcription factor (TF) scans the DNA grooves for hydrogen bonding opportunities in a pre-aligned orientation with occasional slippage out of the groove.

biophysics↗