Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

Summary

We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes.

Abstract

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the transcription termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a transcriptionally active polymerase beyond the 3′ end of the gene as an indicator of a transcription termination defect¹. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the termination signal is the one that initiated transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3′ end of the gene. Secondly, it can distinguish if the transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene².

Introduction

The eukaryotic transcription cycle consists of three major steps; initiation, elongation and termination. The termination of transcription by RNA polymerase II consists of two distinct, interdependent steps³. The first step involves cleavage, polyadenylation and release of mRNA from the template, and is immediately followed by the second step, marked by disengagement of polymerase from the template. Proper termination is crucial for the recycling of the polymerase during initiation/reinitiation of transcription, for preventing interference with the transcription of downstream genes, and for keeping aberrant transcription in check by limiting tr....

Protocol

NOTE: This method was used in the research article reported in Medler, S and Ansari, A.².

1. Culturing and Harvesting the Cells

1. Start a 5 mL culture of cells in YPD medium (10 g/L yeast extract, 20 g/L peptone, 2% dextrose) from a freshly streaked Saccharomyces cerevisiae plate.
2. Grow the cells overnight in an orbital shaker at 30 °C and 250 rpm.
3. Dilute the overnight grown culture 100 times to a final volume of 100 mL in YPD medium in a 250-mL baffled flask.
   NOTE: A total of 100 mL of culture is needed per TRO reaction; if multiple reactions are needed, a larger vol....

Results

To demonstrate the applicability of the BrUTP-strand-specific TRO procedure in detecting a transcription termination defect, we used a termination-defective, temperature-sensitive mutant of RNA14 called rna14-1. The role of Rna14 in termination of transcription by RNA polymerase II was demonstrated using the traditional TRO assay that detected RNA in the terminator-proximal region of selected genes¹. The detection of RNA signal beyond the terminat.......

Discussion

The strand-specific TRO protocol used here was adapted from the protocol used for GRO-Seq (Global Run On-Sequencing) analysis in mammalian cells¹². We successfully modified the protocol to study nascent transcription in budding yeast. To specifically analyze the transcription termination defect, we adjusted the protocol further by getting rid of the RNA hydrolysis step. This allowed us to specifically detect the full length nascent transcripts that initiated from the transcription start site near .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phenol-chloroform-isoamyl alcohol mixture	Sigma-Aldrich	77619	pH 4-5
TRIzol reagent	Life technologies	15596-026
RNase Inhibitor, human placenta	New England Biolabs	M0307S
Anti-BrdU beads	Santa Cruz Biotechnology	sc-32323AC
Protoscript II	New England Biolabs	M03368L	or an equivalent enzyme
Advantage 2 polymerase Mix	Clontech	639201	or an equivalent enzyme
5-Bromouridine 5' triphosphate sodium salt	Santa Cruz Biotechnology	sc-214314A
RNeasy Mini kit	Qiagen	74104
ATP	New England Biolabs	N0451AA
CTP		N0454AA
GTP		N0452AA
Ultra-pure bovine serum albumin (BSA)	MC Labs	UBSA-100
Asc1 B			AGATTCGTCGGTCACAAGTCC
Asc1 C			GAACTTTATACATATTCTTAGTT AGCAGTC
Asc1 D			TGTACATATGTATTTTCGCAGCA
Asc1 E			GCCAAGGAGACTGAATTTAATG
Asc1 F			CTATGGAATGGGGGTTTTAAG
Asc1 G			GGTTATGGCAGACATGCCAC
5s cDNA			AGATTGCAGCACCTGAGT
5’ 5s reverse			GGTTGCGGCCATATCTAC
3’ 5s reverse			TGAGTTTCGCGTATGGTC