In 1965, several laboratories established that RNA synthesis by RNA polymerase occurred in vitro. This discovery revealed an additional factor needed to terminate transcription correctly. A DNA sequence is read by an RNA polymerase which produces a complementary RNA strand called a primary transcript. Only one of the two DNA strands serves as a template for this process. The antisense strand of DNA is read from the 3' end to the 5' end during transcription. The complementary RNA is created in the opposite direction, moving from 5' to 3'. This directionality exists because RNA polymerase can only add nucleotides to the 3' end of the growing mRNA chain. In bacteria, the RNA polymerase core enzyme consists of five subunits: two alpha subunits, one beta subunit, one beta prime subunit, and one omega subunit. It binds to a single transcription factor called the sigma factor. Together they make up the RNA polymerase holoenzyme. Unlike eukaryotes, the initiating nucleotide of nascent bacterial mRNA is not capped with a modified guanine nucleotide. Instead it bears a 5' triphosphate group. In archaea and eukaryotes, RNA polymerase contains subunits homologous to each of the five bacterial RNA polymerase subunits plus unique ones. Archaea have three general transcription factors: TBP, TFB, and TFE. Eukaryotic RNA polymerase II requires six general transcription factors including TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.
Regulatory Networks And Enhancers
Enhancers are regions of the genome that act as major gene-regulatory elements. They control cell-type-specific gene transcription programs by looping through long distances to come in physical proximity with promoters. A study of brain cortical neurons found 24,937 loops bringing enhancers to their target promoters. Multiple enhancers often sit tens or hundreds of thousands of nucleotides distant from their target genes. These elements loop to their target gene promoters and coordinate to control transcription of common targets. An inactive enhancer may be bound by an inactive transcription factor. Phosphorylation of this transcription factor activates it. The activated transcription factor then activates the enhancer to which it is bound. About 60% of promoter sequences have a CpG island while only about 6% of enhancer sequences do. CpG islands constitute regulatory sequences since methylation here can reduce or silence gene transcription. DNA methylation regulates gene transcription through interaction with methyl binding domain proteins like MeCP2, MBD1, and MBD2. These proteins bind most strongly to highly methylated CpG islands. They guide protein complexes with chromatin remodeling activity to these sites. EGR1 protein is a particular transcription factor important for regulation of methylation of CpG islands. There are about 12,000 binding sites for EGR1 in the mammalian genome. Half of these sites are located in promoters and half in enhancers.