Techniques used to study post initiation steps

1.2.2.1 Nuclear Run-on Assay

The run-on transcription assay is the mostly commonly used method for testing

whether a gene is regulated at the transcriptional level. The assay allows the

monitoring of transcriptionally active polymerases by analysis of newly synthesised radiolabelled RNA in the presence of [a -^^P] UTP. Intact mammalian cells are unable to take up nucleoside triphosphates, the run-on transcription reactions are therefore carried out in isolated nuclei. For this purpose the cells are usually lysed with non-ionic detergents such as Nonidet P-40 or Triton X-100 which destroy the cell membrane but leave the nucleus intact. In the first part of this thesis I have used this technique to study transcription in yeast by using yeast cells permeabilised with

Sarkosyl. An advantage to this procedure is that new initiation o f transcription is inhibited and only established transcription com plexes can elongate (Cai and Luse,

1987).

Recent experience, however, indicates that there are possible pitfalls in the interpretation of results obtained by run-on experiments. Polym erases can pause at or im m ediately downstream of the start site of transcription in vivo (Rougvie and Lis, 1988). The mechanisms by which these polymerases are blocked in elongation are not well understood. It is assum ed that paused polym erases close to a prom oter are still part of the initiation complex and have not achieved appropriate signals or factors for form ing an elon g atio n -co m p eten t tran scrip tio n com plex. H o w e v e r , p a u s e d polym erase released artificially from the elongation block during the transcription reaction can give rise to strong run-on transcription activity downstream of the paused site. H ow ever, it is not possible to distinguish if the polym erases are actively transcribing or are stalled when the run-on reaction is done.

Activation o f polym erases paused at or near the transcription start site in the run-on experim ent can result in two m isleading interpretations. 1: If the run on transcripts are probed with a full length cDNA the probe produces a signal w hen transcription is restricted to 5' end of the gene. Thus, the probe produces a signal even when the gene is not completely transcribed. 2; If run-on transcripts are probed w ith shorter probes spanning the 5', middle and 3' end of the gene then an increased signal is produced at the 5' probe com pared to the rest o f the probes. T his can be m isinterpreted to m ean as the gene is tran scrib aid isco n tin u o u sly and th erefo re im plying a block in elongation. However, both interpretationgcan be false and have led to misleading conclusions in the past.

The run-on signal can be seen to be proportional to the density o f polym erases in a particular region o f the gene, averaged over the population o f isolated nuclei in the case if m am malian cells or perm eabilised cells in yeast. The run-on assay does not m easure the initiation rates^ only the distribution of the pre-initiated polym erases (see D iscussion I). However, comparison of the 5' with 3' run-on signals is an inform ative m easure of post-initiation events (prom oter clearance and elongation ) in vivo.

1.2.2.2 Footprinting

This is a widely used technique for identifying a DNA binding site based on the premise that a protein bound tightly to a specific sequence will inhibit or alter access of reagents to the bases or the sugar phosphate backbone of that sequence. Some of the commonly used DNA cleaving or modifying agents include DNasel,

DMS, UV light and KMn0 4.

Footprinting is therefore used to detect the presence of transcription bubbles

with a transcriptionally engaged polymerase for example in the hsp 7 0 genes

(described below). However, a disadvantage of this technique is that signafeare

observed only when all sites are occupied. Moreover, absence of a footprint

does not mean absence of the polymerase at the proposed site but could also mean that polymerases are not stalled at particular position but are scattered uniformly along the gene.

1.2.2.3 RNase protection

It is a transfection technique based on the observation that prematurely terminated HIV RNAs are stable. RNA is isolated from the transfected cells, it is then hybridised to a labelled probe specific to a particular sequence on the gene. Hybridisation is followed by the digestion with RNAse T1 which digests any unhybridised single stranded RNAs. Presence of different lengths of protected RNA fragments indicates if the transcription was proceeding with high efficiency or not. However, one major problem with this technique is that short RNA products which may correspond to premature terminated products are difficult to distinguish from the RNA fragments that result from degradation during the procedure.