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In genetics, transcription is the first of the two-step protein biosynthesis process. Transcription is an enzymatic process that reads DNA and transforms it into its complementary RNA sequence. Transcription ultimately leads to DNA translation.
There is no proofreading or correction process; therefore, it has a lower copying fidelity then DNA replication. It proceeds downstream (5' toward 3').
The transcription process is divided in 3 stages: initiation, elongation and termination.
Prokaryote transcription
- Happens freely in the cytoplasm exactly like in translation.
- Translation can occur even without the transcription completed.
Initiation
The followings steps occur upon initiation:
- The RNAP recognizes the promoter region of the gene and binds to the DNA at that specific location. At this stage, the DNA is still double-stranded and called closed complex.
- Promoter binding is a two step process. Binding is much tighter above 15°C
- The DNA is unwound and becomes single-stranded at the initiation site (the -10 promoter region). This is called open complex.
- The RNA polymerase attempts to transcribe the DNA but produces about 10 abortive transcripts which are unable to leave the RNA polymerase because the exit channel is blocked by the σ-factor.
- At some point, the σ-factor dissociates from the holoenzyme, and elongation continues.
RNA polymerase prefers to start transcripts with ATP, and to a lesser extent GTP (purine nucleotide triphosphates). UTP, and CTP are disfavoured (pyrimidine nucleotide triphosphates).
Regulation
Selective transcription is mainly responsible for the differential protein synthesis among various types of cells in the same organism.
Elongation
The RNA polymerase runs along the DNA, synthesizing mRNA in the process. In bacteria, the nascending mRNA is processed right away by ribosomes.
Termination
Two termination mechanisms are well known:
- Intrinsic termination involves terminator sequences within the RNA as it is being made that signal the RNA polymerase to stop. The terminator sequence is usually a palindromic DNA sequence that forms a hairpin.
- Rho-dependent termination uses a termination factor called ρ factor to stop RNA synthesis at specific sites. This protein binds and runs along the mRNA towards the RNAP. When ρ-factor reaches the RNAP, it causes RNAP to dissociate from the DNA, terminating transcription.
Other termination mechanisms include the fact that transcription will terminate if the RNAP comes across a region with repetitious base pairs (for example, TTTTTT).
A (simple) model for a bacterial gene to be transcribed looks like this:
upstream [[promoter]] downstream
5'--- |-35|----//-----|-10|-------------------------------------------|T|------------3' (Message/Non-Template Strand)
| "+1" site of initiation
--------------------->
mRNA
where the -35 and -10 region base pairs is called the promoter, and |T| stands for terminator. The DNA on the template strand between the +1 site and the terminator is transcribed to mRNA, which is then translated into protein.
Promoters can differ in strength, that is, how attractive they are for RNA polymerase. The more similar they are to a consensus sequence, the stronger they are. The "ideal" promoter in E. coli looks like this:
5'----TTGACA---|17 bp|----TATAAT---|7bp|---|purines|----3'
Eukaryote transcription
The basal transcription complex includes the RNA polymerase and additional proteins that are necessary for correct initiation and elongation of RNA synthesis. Eukaryotes have evolved more complex regulatory mechanisms than prokaryotes. For instance, in eukaryotes the genetic material (DNA) is synthesized in the nucleus, separated from the site of translation, the cytoplasm, by the nuclear membrane. This allows temporal regulation of gene expression by sequestration of the RNA in the nucleus, and allows for selective transport of RNAs to the cytoplasm, where the ribosome resides. Primary transcripts in eukaryotic cells are also synthesized as a larger precursor RNAs that must be processed by splicing out introns (non-coding sequences) and ligating exons (non-contiguous coding sequences) into the mature mRNA. Primary transcripts for some genes can be quite large. The primary transcripts of the neurexin genes, for instance, are as large as 1.7 megabases (1,700,000 bases), while the mature neurexin mRNAs are under 10 kilobases (10,000 bases), with as many as 24 exons and thousands of possible alternative splice variants that produce proteins with different activities.
Gene expression in eukaryotes is also controlled by complex interactions between cis-acting sites within the regulatory regions of the DNA, and trans-acting factors that include transcription factors and the basal transcription complex, but eukaryotes have evolved a much more complex system for regulation of transcription. For example, eukaryotes have three RNA polymerases, in contrast to prokaryotes, which only have one.
- RNA Polymerase I is located in the nucleolus and transcribes only rRNAs.
- RNA Polymerase II transcribes messenger RNA.
- RNA Polymerase III transcribes tRNAs and other small RNAs.
The C-terminus of all three RNA polymerases is highly conserved and binds to two enzyme factors which sense a poly-adenylation sequence. These factors bind to the DNA and attach approximately 200 adenines to the 3' end of the mRNA.
Initiation
The core promoter of eukaryotic genes, where the core transcription complex, including RNA polymerase, is usually a region within 50 bases upstream of the transcription initiation site. Additionally, there can be an upstream control element usually present within 2000 bases upstream of the transcription initiation site. Some genes use enhancer elements that can be thousands of bases upstream or downstream of the transcription initiation site. Combinations of these upstream elements regulate and amplify the formation of the basal transcription complex. This UCE usually contains a TATA box, a highly conserved DNA sequence that reads
- T A T A T/A A
A similar sequence, thus not that highly conserved, is found in the INR element (initiator element, part of the complex core promoter).
Measuring and detecting transcription
Transcription can be measured and detected in a variety of ways:
- Northern hybridization
- RNase protection assay
- Reverse transcriptase-polymerase chain reaction (RT-PCR)
- In vitro transcription
- In situ hybridization
History
RNA synthesis by RNA polymerase had been established in vitro by several laboratories by 1965, however the RNA synthesized by these enzymes had properties that suggested the existence of an additional factor needed to terminate transcription correctly.
By the late 1960s several papers that came out of the Harvard University Biological Laboratories established the basic mechanics of gene expression in bacteria.
See also
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