In this article we will discuss about the role of RNA in transcription regulation.
In the early 1960s, Francois Jocob and Jaques Monod performed exemplary work pertaining to prokaryotic gene regulation. These experimental techniques and strategies were later extended to eukaryotic system.
Although control of transcription between prokaryotic and eukaryotic genes shares substantial homology, eukaryotic control of system is highly complex and deserves greater degree of attention with the advancement of latest technology, understanding eukaryotic transcription regulations have been well documented.
In eukaryotes, three RNA polymerase complexes play important role in transcription. As a general rule, RNA polymerase I transcribe ribosomal RNAs, RNA polymerase II transcribe protein encoding genes and RNA polymerase III transcribe tRNA genes. The RNA polymerase II basal transcription complex consists of RNA polymerase II subunits with general transcription factors such as TBP (Tata Binding Proteins) and TFIIB.
This complex in association with promoter sequence is in close proximity to the transcription initiation. In addition, other regulatory sequences which can positively influence transcription processes are activator or enhancer sequences. Traditionally, enhancers were identified to execute their function at different distances relative to the transcription sites.
In vitro Transcription Assays:
Experiments on transcriptional regulation have been advanced by the use of reagents such as recombinant protein, transcriptionally active cell extract factors, antibodies, and reporter vectors. Widespread utilization of in vitro transcription assays identified specific factors and enzymes in transcription initiation. These test tube assays described to design to study RNA polymerase II dependent transcription.
Whole cell and nuclear extract can support RNA polymerase II dependent transcription. In animals, HeLa nuclear extract is well characterised cell-free system for in vitro transcription studies of human and mammalian promoters. However, extract prepared from Drosophila embyros are more effective than HeLa nuclear extract.
Run-off transcription assays a novel method in which template DNA is incubated with nuclear extract in the presence of exogenously added ribonucleotides (one of which is labelled). After the reaction, RNA transcripts are separated by gel electrophoresis. These assays are utilised to perform and optimise the reactions.
Primer Extension Analysis:
Quantification of the amount of a particular transcript and determination of location of the 5′-end of specific RNAs play significant role in transcription regulation. This process could be accomplished by primer extension analysis in vitro. In this reaction, an end-labelled oligonucleotide is hybridized to non-radioactive run-off RNA. This serves as primer for reverse transcription.
The length of cDNA reflects the number of bases between the labelled nucleotide of the primer and the 5′-end of the RNA and quantity of cDNA product is proportional to the amount of targeted RNA. Primer extension analysis not only detects the specific RNA transcript of interest but also used to map multiple transcription start sites (Fig. 13.16).
