Journal: iScience

Article Title: Maintenance of a host-specific minority mutation in the West Nile virus NS3

doi: 10.1016/j.isci.2023.107468

Figure Lengend Snippet:

Article Snippet: α 32 P-ATP , Perkin Elmer , Cat# BLU003H250UC.

Techniques: Mutagenesis, Recombinant, Transfection, Sequencing, Plasmid Preparation, Software

Journal: Nature

Article Title: Tracking transcription–translation coupling in real time

doi: 10.1038/s41586-024-08308-w

Figure Lengend Snippet: Single-round transcription assays with assembled 70S collided expressome using prNQ215 DNA template. Stalled transcription elongation complex (stalled TEC) was formed with 50 nM DNA template, 200 nM E. coli RNAP, 100 µM ACU trinucleotide, 5 µM GTP and 5 µM ATP (+150 nM 32 P α-ATP) halting RNAP initially at U24 to prevent loading of multiple RNAPs. Then RNAP was walked to desired stalling site by addition of 10 µM UTP and simultaneous addition of 10 µg/mL rifampicin (to prevent transcription re-initiation). 70S PIC was formed on the stalled TEC in presence of 2 µM IF2a, 1 µM fmet-tRNA fmet and 4 mM GTP. This stalled expressome was chased in presence or absence of NusA and/or NusG with 50 µM NTPs (each) at room temperature and per condition time points were taken at 0, 10, 20, 30, 40, 60, 90, 120, 180, 240, 360 and 600 s. Stalled expressome band (91 nt) was immediately chased after NTP addition (<10 s). The gels stem from one replicate.

Article Snippet: In brief, 50 nM DNA template was incubated (20 min, 37 °C) with four equivalents of RNAP in the presence of 100 µM ACU trinucleotide, 5 µM GTP, 5 µM ATP (+150–300 nM 32 P α-ATP, Hartmann Analytic), halting the polymerase at U24, to prevent loading of multiple RNAPs on the same DNA template.

Techniques:

Journal: Nature

Article Title: Tracking transcription–translation coupling in real time

doi: 10.1038/s41586-024-08308-w

Figure Lengend Snippet: a , Overlay of expressome structures (6ztn, 6x7f, 6xdq, 6x6t, 6vu3, 6zto) , showing the variation of FRET distances between the ribosomal h33a (16S rRNA) and RNAP β’ (Nter=black and Cter=pink) labeling sites. Structures were aligned on the RNAP. Helix 33 is color coded according to pdb ID and displayed as cartoon representation. Labeling sites on RNAP and 16S rRNA are indicated as spheres. In all displayed coupled states, the labeling sites are in FRET distance, whereas in the collided state and uncoupled state, they are too far to be detected by FRET (>100 Å). b , Introduction of the ybbR-peptide tag as well as the Cy5 label do not significantly affect RNAP activity: RNAP-Cy5 activity test using single-round transcription assays. Area of total RNA (mean of duplicates) was integrated and normalized to WT RNAP. Individual data points are shown. c , d , Overview of all pdb-deposited expressome structures , (n = 37), which serve as the structural basis for the ribosome-RNAP ( c ) or ribosome-DNA ( d ) FRET signal. Distances are plotted as a function of the intervening mRNA illustrating that our FRET signal is specific for the coupled states. Collided state structures are shown in red, coupled state structures are shown in green and uncoupled state structures are shown in gray. For ( c ), distances were measured between E16 C-alpha (RNAP β’) and U1025 C3’ (16S rRNA) and for ( d ), the distances were measured between the same residue of RNAP and DNA 6 nt downstream from the active site to the non-template strand, where the Cy3.5 DNA label is located during transcription termination. Distances to alternative 30S/RNAP complexes relevant to translation initiation rather than elongation , were also evaluated (>130 Å) but cannot be plotted here, as they lack nucleic acids. Some uncoupled structures (6ztp, 6zto cluster 4 and 5) also fall in FRET range, however those structures were only obtained at a shorter 38 nt intervening mRNA length and were not observed at longer mRNA lengths used in our study to track ribosome/RNAP coupling. Vertical dashed line in plots at 46 nt represents the shortest construct used for the coupled state in this study.

Article Snippet: In brief, 50 nM DNA template was incubated (20 min, 37 °C) with four equivalents of RNAP in the presence of 100 μM ACU trinucleotide, 5 μM GTP, 5 μM ATP (+150–300 nM 32 P α-ATP, Hartmann Analytic), halting the polymerase at U24, to prevent loading of multiple RNAPs on the same DNA template.

Techniques: Labeling, Activity Assay, Residue, Construct

Journal: Nature

Article Title: Tracking transcription–translation coupling in real time

doi: 10.1038/s41586-024-08308-w

Figure Lengend Snippet: a , DNA template design. T7te, T7 terminator. b , Location of labelling sites within coupled expressome (PDB: 6XDQ ). c , Total population of coupled states (loosely coupled and coupled) in dependence of intervening mRNA length. Mean of two replicates is shown as black line. Number of analysed molecules ( n ) and data used are the same as depicted in f . d , e , Schematic (left) and representative smoothed traces (right) for coupled ( d ) and uncoupled ( e ) expressomes. f , FRET distribution histograms with varying mRNA length. Uncoupled ( E FRET ≈ 0), loosely coupled ( E FRET ≈ 0.1) and coupled states ( E FRET ≈ 0.3) are indicated. Number of analysed molecules ( n ) is indicated. Data were combined from two replicates. g , FRET distribution histograms with different Nus factor compositions for mRNA-85. Number of analysed molecules ( n ) is indicated. Data were combined from two replicates. FRET distribution histogram shown for mRNA-85 without factors is the same as shown in f . NusAG, NusA plus NusG.

Article Snippet: In brief, 50 nM DNA template was incubated (20 min, 37 °C) with four equivalents of RNAP in the presence of 100 μM ACU trinucleotide, 5 μM GTP, 5 μM ATP (+150–300 nM 32 P α-ATP, Hartmann Analytic), halting the polymerase at U24, to prevent loading of multiple RNAPs on the same DNA template.

Techniques:

Journal: Nature

Article Title: Tracking transcription–translation coupling in real time

doi: 10.1038/s41586-024-08308-w

Figure Lengend Snippet: Representative smoothed traces for transcription out of coupled ( a-c ) or collided ( d ) state. Data was acquired with alternative laser excitation at wavelengths of 532 nm and 638 nm. The reaction was started with delivery of 50 µM of each NTP. The 70S ribosome was kept stalled on the RBS. a , Illustration of single-molecule trace, where both machines remain coupled throughout the complete transcription reaction. The steady increase in Cy3.5-Cy5 FRET efficiency towards the end of transcription (at ~160–170 s) directly shows active transcription elongation while both machines are coupled containing an intervening mRNA length of 156 nt. b , Single-molecule trace (smoothed) with photobleaching of the RNAP-Cy5 before transcription is completed. Time-evolution of coupling cannot be tracked by 30S-Cy3 and RNAP-Cy5 FRET anymore. Moreover, the expressome uncouples after RNAP-photobleaching and before transcription end, as also no 30S-Cy3 to DNA-Cy3.5 FRET is detected (in contrast to Fig. ). c, d , Assignment of 3-color data by thresholding exemplified on representative, smoothed traces. Thresholds for 30S-RNAP FRET transitions are shown as red horizontal dashed lines, which allows for accurate determination of the coupled dwell times, and thresholds for 30S-DNA ( c ) or DNA-RNAP ( d ) FRET transitions are displayed as yellow orange horizontal dashed lines, which allows for the accurate determination of transcription end (see black arrows). Green-yellow arrows depict bleedthrough from green Cy3-channel to yellow-Cy3.5 channel and yellow-red arrows depict bleedthrough from yellow-Cy3.5 channel to red-Cy5 channel.

Article Snippet: In brief, 50 nM DNA template was incubated (20 min, 37 °C) with four equivalents of RNAP in the presence of 100 μM ACU trinucleotide, 5 μM GTP, 5 μM ATP (+150–300 nM 32 P α-ATP, Hartmann Analytic), halting the polymerase at U24, to prevent loading of multiple RNAPs on the same DNA template.

Techniques:

Journal: Nature

Article Title: Tracking transcription–translation coupling in real time

doi: 10.1038/s41586-024-08308-w

Figure Lengend Snippet: a-d , Single-round transcription assays with coupled expressome using the prNQ216 DNA template. h101 of the 50S mutant was pre-annealed with a biotin-oligonucleotide and the formed expressomes were purified using streptavidin beads to enrich for fully assembled transcription-translation complexes. The purified stalled expressome (+70S) or the stalled transcription complex alone (−70S) were chased in presence or absence of Nus factors (w/o factors, w/ NusA/NusG, w/ NusA only, w/ NusG only; 1 µM final concentration for each Nus factor) and with 50 µM NTP (each) at room temperature. For each condition, time points were taken at 0, 10, 20, 30, 40, 60, 90, 120, 180, 240, 360 and 600 s. e , Pause-escape lifetimes for pause 1 and pause 2 in presence of NusA and in presence (orange) or absence (blue) of ribosome. Natural logarithm of normalized band intensities (P/T) was plotted as function of time and pause-escape lifetimes were fitted with a linear fit function (y = m*x + b, with m being the rate constant) . Data range that was used for fitting is indicated with arrows. Pause-escape lifetime (τ) errors were obtained by error propagation of linear least square fit error for the rate constant. f , Ribosome can activate RNAP only when sharing the same mRNA: Single-round transcription assays with mRNA-46 in presence of NusA and with 1 µM 70S ( in trans , loaded on 6(FK) mRNA) analyzed by denaturing gel electrophoresis. Stalled TEC, pause 1, pause 2 and full-length mRNA bands are shown. The gel stems from one replicate. g , Normalized band intensities (P/T) from gels shown in panels a-d are displayed as a function of time. The displayed datapoints are from 2 biological replicates. Bands for pause 1, pause 2 and full-length (FL) RNA were integrated and divided by the total RNA per lane . Parts of panels ( b ), ( d ) and ( g ) are also shown in main Fig. . h , Secondary structure prediction of the nascent mRNA (mRNA-46) using the RNA structure web server ( https://rna.urmc.rochester.edu/RNAstructureWeb/ ). Top prediction shows secondary structure at pause site 1 (134 nt) and bottom prediction shows secondary structure at pause site 2 (169 nt). The secondary structure was predicted using default settings on the website for 21 °C, forcing the ribosome binding site (as it is masked by the ribosome) and 12 nt (ref. ) upstream from 3’-end of the nascent mRNA (as they are masked by the paused RNAP) to be single-stranded. Color code corresponds to probability of base-pair formation. Position of the ribosome and the RNAP are indicated. Start codon (AUG) is highlighted with a black box.

Article Snippet: In brief, 50 nM DNA template was incubated (20 min, 37 °C) with four equivalents of RNAP in the presence of 100 μM ACU trinucleotide, 5 μM GTP, 5 μM ATP (+150–300 nM 32 P α-ATP, Hartmann Analytic), halting the polymerase at U24, to prevent loading of multiple RNAPs on the same DNA template.

Techniques: Mutagenesis, Purification, Concentration Assay, Nucleic Acid Electrophoresis, Binding Assay

Journal: Nature

Article Title: Tracking transcription–translation coupling in real time

doi: 10.1038/s41586-024-08308-w

Figure Lengend Snippet: Single-round transcription assays with assembled 70S collided expressome using prNQ215 DNA template. Stalled transcription elongation complex (stalled TEC) was formed with 50 nM DNA template, 200 nM E. coli RNAP, 100 µM ACU trinucleotide, 5 µM GTP and 5 µM ATP (+150 nM 32 P α-ATP) halting RNAP initially at U24 to prevent loading of multiple RNAPs. Then RNAP was walked to desired stalling site by addition of 10 µM UTP and simultaneous addition of 10 µg/mL rifampicin (to prevent transcription re-initiation). 70S PIC was formed on the stalled TEC in presence of 2 µM IF2a, 1 µM fmet-tRNA fmet and 4 mM GTP. This stalled expressome was chased in presence or absence of NusA and/or NusG with 50 µM NTPs (each) at room temperature and per condition time points were taken at 0, 10, 20, 30, 40, 60, 90, 120, 180, 240, 360 and 600 s. Stalled expressome band (91 nt) was immediately chased after NTP addition (<10 s). The gels stem from one replicate.

Article Snippet: In brief, 50 nM DNA template was incubated (20 min, 37 °C) with four equivalents of RNAP in the presence of 100 μM ACU trinucleotide, 5 μM GTP, 5 μM ATP (+150–300 nM 32 P α-ATP, Hartmann Analytic), halting the polymerase at U24, to prevent loading of multiple RNAPs on the same DNA template.

Techniques: