Atomic Force Microscopy Imaging and Force Spectroscopyof RNA

Bussiek, Malte; Schöne, Antonie; Nellen, Wolfgang

doi:10.1002/9783527647064.ch25

Cited by 2 publications

(1 citation statement)

References 31 publications

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“…Before the actual measurements, the density of molecules was recorded in a test image; depending on the observed density, RNA mixtures were diluted with 1 × TE buffer to adjust the working concentrations to ~1-4 nM. RNA solutions were transferred to a mica surface treated with poly-L-lysine (MW 500-2000, Sigma-Aldrich, Taufkirchen, Germany) as described [26]. Pictures were taken using a Veeco Multimode Nanoscope IIIa AFM device with silicon tips (Tap300Al-G, BudgetSensors, Sofia, Bulgaria) in tapping mode (resonance frequency: 300 kHz, scan rate: 1 Hz).…”

Section: Atomic Force Microscopy (Afm)mentioning

confidence: 99%

Structural and Functional Insight into the Mechanism of Bacillus subtilis 6S-1 RNA Release from RNA Polymerase

Ganapathy

Hoch

Lechner

et al. 2022

ncRNA

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Here we investigated the refolding of Bacillus subtilis 6S-1 RNA and its release from σA-RNA polymerase (σA-RNAP) in vitro using truncated and mutated 6S-1 RNA variants. Truncated 6S-1 RNAs, only consisting of the central bubble (CB) flanked by two short helical arms, can still traverse the mechanistic 6S RNA cycle in vitro despite ~10-fold reduced σA-RNAP affinity. This indicates that the RNA’s extended helical arms including the ‘−35′-like region are not required for basic 6S-1 RNA functionality. The role of the ‘central bubble collapse helix’ (CBCH) in pRNA-induced refolding and release of 6S-1 RNA from σA-RNAP was studied by stabilizing mutations. This also revealed base identities in the 5’-part of the CB (5’-CB), upstream of the pRNA transcription start site (nt 40), that impact ground state binding of 6S-1 RNA to σA-RNAP. Stabilization of the CBCH by the C44/45 double mutation shifted the pRNA length pattern to shorter pRNAs and, combined with a weakened P2 helix, resulted in more effective release from RNAP. We conclude that formation of the CBCH supports pRNA-induced 6S-1 RNA refolding and release. Our mutational analysis also unveiled that formation of a second short hairpin in the 3′-CB is detrimental to 6S-1 RNA release. Furthermore, an LNA mimic of a pRNA as short as 6 nt, when annealed to 6S-1 RNA, retarded the RNA’s gel mobility and interfered with σA-RNAP binding. This effect incrementally increased with pLNA 7- and 8-mers, suggesting that restricted conformational flexibility introduced into the 5’-CB by base pairing with pRNAs prevents 6S-1 RNA from adopting an elongated shape. Accordingly, atomic force microscopy of free 6S-1 RNA versus 6S-1:pLNA 8- and 14-mer complexes revealed that 6S-1:pRNA hybrid structures, on average, adopt a more compact structure than 6S-1 RNA alone. Overall, our findings also illustrate that the wild-type 6S-1 RNA sequence and structure ensures an optimal balance of the different functional aspects involved in the mechanistic cycle of 6S-1 RNA.

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Section: Atomic Force Microscopy (Afm)mentioning

confidence: 99%

Structural and Functional Insight into the Mechanism of Bacillus subtilis 6S-1 RNA Release from RNA Polymerase

Ganapathy

Hoch

Lechner

et al. 2022

ncRNA

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Investigation of HIV-1 Gag binding with RNAs and lipids using Atomic Force Microscopy

Chen

Liu

et al. 2020

PLoS ONE

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Atomic Force Microscopy was utilized to study the morphology of Gag, ΨRNA, and their binding complexes with lipids in a solution environment with 0.1Å vertical and 1nm lateral resolution. TARpolyA RNA was used as a RNA control. The lipid used was phospha-tidylinositol-(4,5)-bisphosphate (PI(4,5)P2).The morphology of specific complexes Gag-ΨRNA, Gag-TARpolyA RNA, Gag-PI(4,5)P2 and PI(4,5)P2-ΨRNA-Gag were studied. They were imaged on either positively or negatively charged mica substrates depending on the net charges carried. Gag and its complexes consist of monomers, dimers and tetramers, which was confirmed by gel electrophoresis. The addition of specific ΨRNA to Gag is found to increase Gag multimerization. Non-specific TARpolyA RNA was found not to lead to an increase in Gag multimerization. The addition PI(4,5)P2 to Gag increases Gag multimerization, but to a lesser extent than ΨRNA. When both ΨRNA and PI(4,5)P2 are present Gag undergoes comformational changes and an even higher degree of multimerization.

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Atomic Force Microscopy Imaging and Force Spectroscopyof RNA

Cited by 2 publications

References 31 publications

Structural and Functional Insight into the Mechanism of Bacillus subtilis 6S-1 RNA Release from RNA Polymerase

Structural and Functional Insight into the Mechanism of Bacillus subtilis 6S-1 RNA Release from RNA Polymerase

Investigation of HIV-1 Gag binding with RNAs and lipids using Atomic Force Microscopy

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