Journal: Cell

Article Title: Transient protein-RNA interactions guide nascent ribosomal RNA folding

doi: 10.1016/j.cell.2019.10.035

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains E. coli BL21-Gold (DE3) Agilent Cat#230132 E. coli Tuner ™ (DE3) Novagen Cat#70623-3 Chemicals, Peptides, and Recombinant Proteins Protocatechuic acid (PCA) Pacific Biosciences Cat#100-215-400 Protocatechuate-3,4-dioxygenase (PCD) Pacific Biosciences Cat#001-028-310 TSY Pacific Biosciences Cat#100-214-900 Cy5 Maleimide Mono-Reactive Dye 5-Pack GE Healthcare Cat#PA25031 Cy5.5 Sulfo-Cyanine5 maleimide Lumiprobe Cat#13380 Cy3 Maleimide Mono-Reactive Dye 5-Pack GE Healthcare Cat#PA23031 E. coli RNA polymerase holoenzyme NEB Cat#M0551S Biolipidure-203 NOF America Corporation Cat#Biolipidure-203 Biolipidure-206 NOF America Corporation Cat#Biolipidure-206 Purified ribosomal proteins of 16S rRNA 3’domain This paper N/A Oligonucleotides Primers, geneBlocks-based DNA transcription templates, labeled DNA oligonucleotides See Data S1 N/A ACU RNA trinucleotide Dharmacon N/A Recombinant DNA pRSF-1b Novagen Cat#71330-3 pRSF-1b-S2 This paper Addgene Plasmid #128590 pRSF-1b-S3 This paper Addgene Plasmid #128591 pRSF-1b-S7-S83C/delta157-178 This paper Addgene Plasmid #128592 pRSF-1b-S10 This paper Addgene Plasmid #128594 pRSF-1b-S13-C85S/P112C This paper Addgene Plasmid #128595 pRSF-1b-S14 This paper Addgene Plasmid #128596 pRSF-1b-S19-T48C This paper Addgene Plasmid #128597 pET-S9 This paper Addgene Plasmid #132948 pRSF-1b-S3-M129C This paper Addgene Plasmid #132949 pRSF-1b-S15-T79C This paper Addgene Plasmid #133048 Software and Algorithms MATLAB R2015a Matworks Inc http://matlab.com Open in a separate window KEY RESOURCES TABLE

Techniques: Recombinant, Purification, Labeling, Plasmid Preparation, Software

Journal: Cell

Article Title: Transient protein-RNA interactions guide nascent ribosomal RNA folding

doi: 10.1016/j.cell.2019.10.035

Figure Lengend Snippet: (A) 3-dimensional structure of E. coli 30S ribosomal subunit with labeling sites for Cy5-S3 and Cy5.5-S7 (PDB accession code: 4V9P); the Cy3 dye of the labeled DNA oligonucleotide binding to the 3’-end of the nascent RNA is modeled into the structure. (B) Experimental setup: 100 nM Cy5-S3, 5 nM Cy5.5-S7 and 400 nM of all the other unlabeled 3’domain r-proteins were injected; 35 °C. (C) Representative single-molecule trace with Cy5-S3 (red), Cy5.5-S7 (purple) and Cy3-oligo (green). The trace was not corrected for fluorescence spectral bleed-through from the Cy5 into the Cy5.5 channel and the reverse. (D) The first Cy5-S3 binding event always follows the final Cy5.5-S7 binding event demonstrating sequential ribosome assembly. (E) The assembly process from stable S7 incorporation until 3’domain completion is slow, indicating that RNA conformational changes are also required later in assembly. Number of molecules analyzed in (D and E) (n) = 64. See also Figure S7 and Data S2.

Article Snippet: Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains E. coli BL21-Gold (DE3) Agilent Cat#230132 E. coli Tuner ™ (DE3) Novagen Cat#70623-3 Chemicals, Peptides, and Recombinant Proteins Protocatechuic acid (PCA) Pacific Biosciences Cat#100-215-400 Protocatechuate-3,4-dioxygenase (PCD) Pacific Biosciences Cat#001-028-310 TSY Pacific Biosciences Cat#100-214-900 Cy5 Maleimide Mono-Reactive Dye 5-Pack GE Healthcare Cat#PA25031 Cy5.5 Sulfo-Cyanine5 maleimide Lumiprobe Cat#13380 Cy3 Maleimide Mono-Reactive Dye 5-Pack GE Healthcare Cat#PA23031 E. coli RNA polymerase holoenzyme NEB Cat#M0551S Biolipidure-203 NOF America Corporation Cat#Biolipidure-203 Biolipidure-206 NOF America Corporation Cat#Biolipidure-206 Purified ribosomal proteins of 16S rRNA 3’domain This paper N/A Oligonucleotides Primers, geneBlocks-based DNA transcription templates, labeled DNA oligonucleotides See Data S1 N/A ACU RNA trinucleotide Dharmacon N/A Recombinant DNA pRSF-1b Novagen Cat#71330-3 pRSF-1b-S2 This paper Addgene Plasmid #128590 pRSF-1b-S3 This paper Addgene Plasmid #128591 pRSF-1b-S7-S83C/delta157-178 This paper Addgene Plasmid #128592 pRSF-1b-S10 This paper Addgene Plasmid #128594 pRSF-1b-S13-C85S/P112C This paper Addgene Plasmid #128595 pRSF-1b-S14 This paper Addgene Plasmid #128596 pRSF-1b-S19-T48C This paper Addgene Plasmid #128597 pET-S9 This paper Addgene Plasmid #132948 pRSF-1b-S3-M129C This paper Addgene Plasmid #132949 pRSF-1b-S15-T79C This paper Addgene Plasmid #133048 Software and Algorithms MATLAB R2015a Matworks Inc http://matlab.com Open in a separate window KEY RESOURCES TABLE

Techniques: Labeling, Binding Assay, Injection, Fluorescence

Journal: Cell

Article Title: Transient protein-RNA interactions guide nascent ribosomal RNA folding

doi: 10.1016/j.cell.2019.10.035

Figure Lengend Snippet: (A) Secondary structure of E. coli S7 binding site. (B) 3-dimensional structure of E. coli 30S ribosomal subunit with labeling sites (PDB accession code: 4V9P); the Cy3 dye of the labeled DNA oligonucleotide binding to the 3’-end of the nascent RNA is modeled into the structure. (C and D) Single-molecule fluorescence approach (Duss et al., 2018). (E) Representative single-molecule trace with fluorescence intensity (top) and FRET efficiency (bottom); transcription (TC; orange), Cy5-S7 (red), Cy3-oligo (green). See also Figure S1.

Article Snippet: Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains E. coli BL21-Gold (DE3) Agilent Cat#230132 E. coli Tuner ™ (DE3) Novagen Cat#70623-3 Chemicals, Peptides, and Recombinant Proteins Protocatechuic acid (PCA) Pacific Biosciences Cat#100-215-400 Protocatechuate-3,4-dioxygenase (PCD) Pacific Biosciences Cat#001-028-310 TSY Pacific Biosciences Cat#100-214-900 Cy5 Maleimide Mono-Reactive Dye 5-Pack GE Healthcare Cat#PA25031 Cy5.5 Sulfo-Cyanine5 maleimide Lumiprobe Cat#13380 Cy3 Maleimide Mono-Reactive Dye 5-Pack GE Healthcare Cat#PA23031 E. coli RNA polymerase holoenzyme NEB Cat#M0551S Biolipidure-203 NOF America Corporation Cat#Biolipidure-203 Biolipidure-206 NOF America Corporation Cat#Biolipidure-206 Purified ribosomal proteins of 16S rRNA 3’domain This paper N/A Oligonucleotides Primers, geneBlocks-based DNA transcription templates, labeled DNA oligonucleotides See Data S1 N/A ACU RNA trinucleotide Dharmacon N/A Recombinant DNA pRSF-1b Novagen Cat#71330-3 pRSF-1b-S2 This paper Addgene Plasmid #128590 pRSF-1b-S3 This paper Addgene Plasmid #128591 pRSF-1b-S7-S83C/delta157-178 This paper Addgene Plasmid #128592 pRSF-1b-S10 This paper Addgene Plasmid #128594 pRSF-1b-S13-C85S/P112C This paper Addgene Plasmid #128595 pRSF-1b-S14 This paper Addgene Plasmid #128596 pRSF-1b-S19-T48C This paper Addgene Plasmid #128597 pET-S9 This paper Addgene Plasmid #132948 pRSF-1b-S3-M129C This paper Addgene Plasmid #132949 pRSF-1b-S15-T79C This paper Addgene Plasmid #133048 Software and Algorithms MATLAB R2015a Matworks Inc http://matlab.com Open in a separate window KEY RESOURCES TABLE

Techniques: Binding Assay, Labeling, Fluorescence

Journal: Cell

Article Title: Transient protein-RNA interactions guide nascent ribosomal RNA folding

doi: 10.1016/j.cell.2019.10.035

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains E. coli BL21-Gold (DE3) Agilent Cat#230132 E. coli Tuner ™ (DE3) Novagen Cat#70623-3 Chemicals, Peptides, and Recombinant Proteins Protocatechuic acid (PCA) Pacific Biosciences Cat#100-215-400 Protocatechuate-3,4-dioxygenase (PCD) Pacific Biosciences Cat#001-028-310 TSY Pacific Biosciences Cat#100-214-900 Cy5 Maleimide Mono-Reactive Dye 5-Pack GE Healthcare Cat#PA25031 Cy5.5 Sulfo-Cyanine5 maleimide Lumiprobe Cat#13380 Cy3 Maleimide Mono-Reactive Dye 5-Pack GE Healthcare Cat#PA23031 E. coli RNA polymerase holoenzyme NEB Cat#M0551S Biolipidure-203 NOF America Corporation Cat#Biolipidure-203 Biolipidure-206 NOF America Corporation Cat#Biolipidure-206 Purified ribosomal proteins of 16S rRNA 3’domain This paper N/A Oligonucleotides Primers, geneBlocks-based DNA transcription templates, labeled DNA oligonucleotides See Data S1 N/A ACU RNA trinucleotide Dharmacon N/A Recombinant DNA pRSF-1b Novagen Cat#71330-3 pRSF-1b-S2 This paper Addgene Plasmid #128590 pRSF-1b-S3 This paper Addgene Plasmid #128591 pRSF-1b-S7-S83C/delta157-178 This paper Addgene Plasmid #128592 pRSF-1b-S10 This paper Addgene Plasmid #128594 pRSF-1b-S13-C85S/P112C This paper Addgene Plasmid #128595 pRSF-1b-S14 This paper Addgene Plasmid #128596 pRSF-1b-S19-T48C This paper Addgene Plasmid #128597 pET-S9 This paper Addgene Plasmid #132948 pRSF-1b-S3-M129C This paper Addgene Plasmid #132949 pRSF-1b-S15-T79C This paper Addgene Plasmid #133048 Software and Algorithms MATLAB R2015a Matworks Inc http://matlab.com Open in a separate window KEY RESOURCES TABLE

Techniques: Recombinant, Purification, Labeling, Plasmid Preparation, Software