Eukaryotic gene expression
Unlike prokaryotic genes, expression of genes in eukaryotic cells have complex systems of transcription factors that act on promoters to recruit RNA polymerases. Additionally, enhancer elements may reside many kilobase upstream of the promoter. These enhancers strengthen the transcription of the gene. In this case, transcription activator proteins or trans-activators augment the promoter activity. Mediator proteins (coactivators) form a multiprotein complex with the activators to recruit RNA polymerase to the promoter.
Eukaryotic genes may often contain introns (non-coding sequences) that are spliced out from the exons (coding sequences). This complexity permits for increased variety of gene products. Mature eukaryotic mRNAs conatins a 5′-methyl-Guanine followed by an untranslated leader sequence (5′-UTR), the coding sequences (cds), a 3′-untranslated region (3′-UTR) and a long stretch of Adenines (polyA tail).
Expression is most easily measured with RNA since nucleic acid manipulation is fairly simple with 4 different nucleotides. In eukaryotes, the messenger RNA (mRNA) intermediate that is transcribed from DNA contains a polyA tail that is used to separate these messages from other types of RNA that are abundant within cells (like ribosomal RNA). Through the use of an enzyme called reverse transcriptase (RT) and primers composed of deoxy-Thymidines (oligo-dT or dT18), mRNA can be converted into a single strand of DNA that is complimentary to the mRNA. This complimentary DNA is called cDNA. cDNA is very stable compared to the highly labile mRNA and is used for subsequent processing.