Work Description

README for: Single-molecule microscopy image data and analysis files for "A translational riboswitch coordinates nascent transcription-translation coupling." Authors: Surajit Chatterjee, Adrien Chauvier, Shiba S. Dandpat, Irina Artsimovitch and Nils G. Walter Contact: surajitc@umich.edu, achauvie@umich.edu and nwalter@umich.edu Citation to related material: Chatterjee, S., Chauvier, A., Dandpat, S.S., Artsimovitch, I., & Walter, N.G. (2021). A translational riboswitch coordinates nascent transcription-translation coupling. Proc. Natl. Acad. Sci., 118 (16), e2023426118; DOI: 10.1073/pnas.2023426118 Research Overview: These data were generated to study the dynamics of RNAP-ribosome interactions on a nascent mRNA with a preQ1-sensing translational riboswitch in its 5' untranslated region (UTR). Using single molecule fluorescence co-localization, we monitored direct transient binding of 30S ribosomal subunit to the individual nascent mRNA molecules in surface-immobilized paused elongation complexes (PECs). Also, using a novel protein-induced fluorescence enhancement assay we monitored the real-time transcription rate of RNA polymerase (RNAP) under different experimental conditions. The DNA template including the preQ1 riboswitch from B. anthracis under the control of the T7A1 promoter was cloned into pUC19 plasmid. Transcription templates for in vitro transcription were generated by PCR. This work was conducted at the University of Michigan in the Department of Chemistry and was funded by National Institutes of Health (NIH) [GM062357, GM118524 and GM131922 to N.G.W., and grant R01 GM067153 to I.A.] Methodology: Data Collection: Single molecule experiments were performed on a prism-based TIRF microscope (IX 71; Olympus) with a 1.2-numerical aperture 60X water-immersion objective (Olympus UplanApo) and an intensified CCD camera (I-Pentamax, Princeton Instruments). Transient binding of Cy5 labeled 30S subunit to individual Cy3 labeled PEC mRNA molecules immobilized to the surface of the custom-built sample cell was monitored for ~16 min. Both Cy3 and Cy5 dyes were directly and simultaneously excited using 532 nm (green) and 638 nm (red) diode lasers, respectively. Emission from Cy3 and Cy5 were recorded at a 100 ms exposure time. For monitoring real-time transcription rates, the transcription was restarted form a surface immobilized halted complex (HC) at ~10 s of the movie collection by slowly flowing 50 �l imaging solution containing mixture of all four rNTPs. The end of the transcription was characterized by the occurrence of protein-induced fluorescence enhancement (PIFE) signal. Data Analysis: Fluorescence intensity versus time traces for individual molecules ('.traces' files) were first extracted from raw movie files using custom written MATLAB scripts (The MathWorks) in MATLAB R2020a. From the .traces files, genuine single-molecule fluorescence time traces were extracted manually and analyzed using custom scripts written in Matlab and the QuB software suite (State University of New York at Buffalo). Finally, kinetic analyses were performed using hidden-Markov modeling (HMM) with a segmental k-means algorithm as implemented in the program QuB. Analysis of a single movie should take about 1-2 hours depending on the type of experiment. File Organization: This work contains multiple zip files, each of which represents one of the principal experimental conditions presented in the publication. [Name of the zip file Contents] MATLAB scripts Contains all the MATLAB scripts used for single-molecule fluorescence co-localization analysis including kinetic analysis. PEC-99_Cy5-30S Original .traces files, associated files and individual time traces for single-molecule co-localization experiments related to 30S binding to PEC-99 mRNA. PEC-99 + preQ1_Cy5-30S Original .traces files, associated files and individual time traces for single-molecule co-localization experiments related to 30S binding to PEC-99 mRNA in the presence of preQ1. mRNA alone_Cy5-30S Original .traces files, associated files and individual time traces for single-molecule co-localization experiments related to 30S binding to mRNA alone transcript. mRNA alone + preQ1_Cy5-30S Original .traces files, associated files and individual time traces for single-molecule co-localization experiments related to 30S binding to mRNA alone transcript in the presence of preQ1. PEC-99 +ASO_Cy5-30S Original .traces files, associated files and individual time traces for single-molecule co-localization experiments related to 30S binding to PEC-99 mRNA in the presence of ASO blocking strand. PEC-99 +IF mix_Cy5-30S Original .traces files, associated files and individual time traces for single-molecule co-localization experiments related to 30S binding to PEC-99 mRNA in the presence of IF mix. PEC-99 + NusG_Cy5-30S Original .traces files, associated files and individual time traces for single-molecule co-localization experiments related to 30S binding to PEC-99 mRNA in the presence of NusG. PEC-99 + NusG + preQ1_Cy5-30S Original .traces files, associated files and individual time traces for single-molecule co-localization experiments related to 30S binding to PEC-99 mRNA in the presence of NusG and preQ1. PEC-99 + RfaH_Cy5-30S Original .traces files, associated files and individual time traces for single-molecule co-localization experiments related to 30S binding to PEC-99 mRNA in the presence of RfaH. PEC-99 + RfaH + preQ1_Cy5-30S Original .traces files, associated files and individual time traces for single-molecule co-localization experiments related to 30S binding to PEC-99 mRNA in the presence of RfaH and preQ1. real time trx_condition 1 Original .traces files, associated files and individual time traces for real-time transcription rate measurement of RNAP alone. real time trx_condition 2 Original .traces files, associated files and individual time traces for real-time transcription rate measurement of RNAP in the presence of non-translating ribosome. real time trx_condition 3 Original .traces files, associated files and individual time traces for real-time transcription rate measurement of RNAP in the presence of translating ribosome. real time trx with preQ1_condition 1 Original .traces files, associated files and individual time traces for real-time transcription rate measurement of RNAP in the presence of preQ1. real time trx with preQ1_condition 2 Original .traces files, associated files and individual time traces for real-time transcription rate measurement of RNAP in the presence of non-translating ribosome and in the presence of preQ1. real time trx with preQ1_condition 3 Original .traces files, associated files and individual time traces for real-time transcription rate measurement of RNAP in the presence of translating ribosome and in the presence of preQ1. real time trx with chloramphenicol_condition 3 Original .traces files, associated files and individual time traces for real-time transcription rate measurement of RNAP in the presence of translating ribosome and in the presence of translation inhibitor chloramphenicol. real time trx with fusidic acid_condition 3 Original .traces files, associated files and individual time traces for real-time transcription rate measurement of RNAP in the presence of translating ribosome and in the presence of translation inhibitor fusidic acid. anti-SD probe binding kinetics Original .traces files, associated files for measuring anti-SD probe binding kinetics to the full length bas mRNA. Definition of Common Terms and Abbreviations: [Term] [Meaning] UTR untranslated region RNAP RNA polymerase mRNA messenger RNA SD Shine-Dalgarno sequence PEC paused elongation complex HC halted complex PIFE protein-induced fluorescence enhancement HMM hidden-Markov modeling ASO antisense oligonucleotide nt nucleotide