Eukaryotic RNA Polymerases 65
- 20-30 gL Whole-cell or nuclear extract;
- 1-2.5 lag Template DNA;
- 500 I.tM each of ATP, CTP, and UTP;
- 4 mM Creatine phosphate;
- 50 ~tM Unlabeled nucleoside triphosphate (GTP)
- 10 ~tCi Labeled nucleoside triphosphate (32p-GTP specific activity).
The volume is made up to 50 ~L with sterile distilled water and incu-
bated at 30°C for 1 h. Suitable controls include omission of template
DNA or unlabeled nucleoside triphosphates, and addition of RNase or
o~-amanitin.
This system uses template DNA that has been cleaved by a restric-
tion enzyme cutting downstream from its putative start site. RNA
polymerases that transcribe this DNA will stop or "fall off" when they
reach the end of the DNA. If a substantial number of enzymes initiate
transcription at the same site, then a population of molecules will
migrate as a single band during gel electrophoresis. If DNA segments
cleaved by different restriction enzymes are used as templates in sepa-
rate reaction mixtures, the transcription start site can be deduced by
comparison of the sizes of RNAs produced; this can then be used for
promoter mapping.
4.3. Primer Extension
This method is commonly used to measure the precise 5' end of
transcripts, based on the fact that reverse transcriptase can transcribe
RNA into DNA. Therefore, a single-strand oligonucleotide of DNA
primer hybridized to RNA can be extended until the end of the RNA
is reached. Primer extension utilizes a primer of specific sequence to
determine the amount and length of a specific RNA. This sort of analy-
sis has been widely used to characterize mRNA made both in vivo and
in vitro (45).
Primer extension is used to map the 5' termini of an RNA transcript;
hence, the cap site of transcription can be determined. In addition, precur-
sors and processing intermediates of mRNA can be detected. A short
known fragment (30-80 bp) of DNA corresponding to a sequence near to
the anticipated cap site of the gene of interest is labeled at the 5' terminus;
this is termed the primer (a synthetic oligonucleotide or restriction frag-
ment). The test RNA, which has been digested with RNase-free DNase to
remove template DNA, can be synthesized in an in vitro transcription