scholarly journals Single-Molecule Fluorescence Reveals Commonalities and Distinctions among Natural and in Vitro-Selected RNA Tertiary Motifs in a Multistep Folding Pathway

2017 ◽  
Vol 139 (51) ◽  
pp. 18576-18589 ◽  
Author(s):  
Steve Bonilla ◽  
Charles Limouse ◽  
Namita Bisaria ◽  
Magdalena Gebala ◽  
Hideo Mabuchi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Folding Pathway ◽  
Single Molecule Fluorescence

scholarly journals Synthesis runs counter to directional folding of a nascent protein domain

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiuqi Chen ◽  
Nandakumar Rajasekaran ◽  
Kaixian Liu ◽  
Christian M. Kaiser
Keyword(s):  
Single Molecule ◽  
Molten Globule ◽  
Elongation Factor ◽  
Protein Domain ◽  
Folding Pathway ◽  
Native Structure ◽  
Folding Intermediates ◽  
C Terminus

Abstract Folding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.

scholarly journals Synthesis runs counter to directional folding of a nascent protein domain

2020 ◽  
Author(s):  
Xiuqi Chen ◽  
Nandakumar Rajasekaran ◽  
Kaixian Liu ◽  
Christian M. Kaiser
Keyword(s):  
Single Molecule ◽  
Molten Globule ◽  
Elongation Factor ◽  
Protein Domain ◽  
Folding Pathway ◽  
Native Structure ◽  
Folding Intermediates ◽  
C Terminus

AbstractFolding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.

Nucleic acid amplification-integrated single-molecule fluorescence imaging for in vitro and in vivo biosensing

2021 ◽  
Author(s):  
Fei Ma ◽  
Chen-Chen Li ◽  
Chun-Yang Zhang
Keyword(s):  
Nucleic Acid ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
High Sensitivity ◽  
Nucleic Acid Amplification ◽  
Single Molecule Fluorescence

Single-molecule fluorescence imaging is among the most advanced analytical technologies and has been widely adopted for biosensing due to its distinct advantages of simplicity, rapidity, high sensitivity, low sample consumption,...

scholarly journals Light-controlled twister ribozyme with single-molecule detection resolves RNA function in time and space

2020 ◽  
Vol 117 (22) ◽  
pp. 12080-12086 ◽  
Author(s):  
Arthur Korman ◽  
Huabing Sun ◽  
Boyang Hua ◽  
Haozhe Yang ◽  
Joseph N. Capilato ◽  
...  
Keyword(s):  
Single Molecule ◽  
Ground State ◽  
Rna Folding ◽  
High Energy ◽  
Folding Pathway ◽  
Active Conformation ◽  
Single Molecule Fluorescence ◽  
Rapid Control ◽  
Time Observation ◽  
Real Time Observation

Small ribozymes such asOryza sativatwister spontaneously cleave their own RNA when the ribozyme folds into its active conformation. The coupling between twister folding and self-cleavage has been difficult to study, however, because the active ribozyme rapidly converts to product. Here, we describe the synthesis of a photocaged nucleotide that releases guanosine within microseconds upon photosolvolysis with blue light. Application of this tool toO. sativatwister achieved the spatial (75 µm) and temporal (≤30 ms) control required to resolve folding and self-cleavage events when combined with single-molecule fluorescence detection of the ribozyme folding pathway. Real-time observation of single ribozymes after photo-deprotection showed that the precleaved folded state is unstable and quickly unfolds if the RNA does not react. Kinetic analysis showed that Mg2+and Mn2+ions increase ribozyme efficiency by making transitions to the high energy active conformation more probable, rather than by stabilizing the folded ground state or the cleaved product. This tool for light-controlled single RNA folding should offer precise and rapid control of other nucleic acid systems.

scholarly journals Mechanism of broad-spectrum Cas9 inhibition by AcrIIA11

2021 ◽  
Author(s):  
Kaylee E Dillard ◽  
Cynthia Terrace ◽  
Kamyab Javanmardi ◽  
Wantae Kim ◽  
Kevin J Forsberg ◽  
...  
Keyword(s):  
Adaptive Immunity ◽  
Single Molecule ◽  
Broad Spectrum ◽  
Dna Cleavage ◽  
Sequence Similarity ◽  
Mammalian Genome ◽  
Single Molecule Fluorescence ◽  
High Sequence Similarity ◽  
Dna Trapping

Mobile genetic elements evade CRISPR-Cas adaptive immunity by encoding anti-CRISPR proteins (Acrs). Acrs inactivate CRISPR-Cas systems via diverse mechanisms but are generally specific for a narrow subset of Cas nucleases that share high sequence similarity. Here, we demonstrate that AcrIIA11 inhibits diverse Cas9 sub-types in vitro and human cells. Single-molecule fluorescence imaging reveals that AcrIIA11 interferes with the first steps of target search by reducing S. aureus Cas9′s diffusion on non-specific DNA. DNA cleavage is inhibited because the AcrIIA11:Cas9 complex is kinetically trapped at PAM-rich decoy sites, preventing Cas9 from reaching its target. This work establishes that DNA trapping can be used to inhibit a broad spectrum of Cas9 orthologs in vitro and during mammalian genome editing.

Biology on track: single-molecule visualisation of protein dynamics on linear DNA substrates

2020 ◽  
Author(s):  
Gurleen Kaur ◽  
Lisanne M. Spenkelink
Keyword(s):  
Single Molecule ◽  
Molecular Motor ◽  
Imaging Techniques ◽  
Biological Research ◽  
Dna Interactions ◽  
Single Molecule Fluorescence ◽  
Linear Dna ◽  
Protein Dna Interactions ◽  
Dna Substrates

Abstract Single-molecule fluorescence imaging techniques have become important tools in biological research to gain mechanistic insights into cellular processes. These tools provide unique access to the dynamic and stochastic behaviour of biomolecules. Single-molecule tools are ideally suited to study protein–DNA interactions in reactions reconstituted from purified proteins. The use of linear DNA substrates allows for the study of protein–DNA interactions with observation of the movement and behaviour of DNA-translocating proteins over long distances. Single-molecule studies using long linear DNA substrates have revealed unanticipated insights on the dynamics of multi-protein systems. In this review, we provide an overview of recent methodological advances, including the construction of linear DNA substrates. We highlight the versatility of these substrates by describing their application in different single-molecule fluorescence techniques, with a focus on in vitro reconstituted systems. We discuss insights from key experiments on DNA curtains, DNA-based molecular motor proteins, and multi-protein systems acting on DNA that relied on the use of long linear substrates and single-molecule visualisation. The quality and customisability of linear DNA substrates now allows the insertion of modifications, such as nucleosomes, to create conditions mimicking physiologically relevant crowding and complexity. Furthermore, the current technologies will allow future studies on the real-time visualisation of the interfaces between DNA maintenance processes such as replication and transcription.

scholarly journals Mechanisms that ensure speed and fidelity in eukaryotic translation termination

2021 ◽  
Author(s):  
Michael R Lawson ◽  
Laura N Lessen ◽  
Jinfan Wang ◽  
Arjun Prabhakar ◽  
Nicholas C Corsepius ◽  
...  
Keyword(s):  
Single Molecule ◽  
Translation Termination ◽  
Translation System ◽  
Single Molecule Fluorescence ◽  
Translation Elongation ◽  
Eukaryotic Translation ◽  
Stop Codons ◽  
Release Factors ◽  
Molecular Events

Translation termination, which liberates a nascent polypeptide from the ribosome specifically at stop codons, must occur accurately and rapidly. We established single molecule fluorescence assays to track the dynamics of ribosomes and two requisite release factors (eRF1 and eRF3) throughout termination using an in vitro reconstituted yeast translation system. We found that the two eukaryotic release factors bind together to recognize stop codons rapidly and elicit termination via a tightly regulated, multi-step process that resembles tRNA selection during translation elongation. Because the release factors are conserved from yeast to humans, the molecular events that underlie yeast translation termination are likely broadly fundamental to eukaryotic protein synthesis.

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