Transcription

A micrograph of ongoing gene transcription of ribosomal RNA illustrating the growing primary transcripts. "Begin" indicates the 3' end of the DNA template strand, where new RNA synthesis begins; "end" indicates the 5' end, where the primary transcripts are almost complete.

As in DNA replication, transcription proceeds in the 5' → 3' direction (i.e. the old polymer is read in the 3' → 5' direction and the new, complementary fragments are generated in the 5' → 3' direction). In the case of transcription, the "old polymer" is the DNA template (non-coding) strand. RNA polymerase binds to the 3' end of a gene on the DNA template strand and travels toward the 5' end. In the process, the RNA polymerase synthesizes an RNA molecule from its 5' end to the 3' end. Except for the fact that thymines in DNA are converted to uracils in RNA, the newly synthesized RNA strand will have the same sequence as the coding (non-template) strand of the DNA. For this reason, scientists usually refer to the DNA coding strand when referring to the directionality of genes on DNA, not the template strand. Thus, genes are said to be transcribed in the 5' → 3' direction.

Transcription is divided into 3 stages: initiation, elongation and termination.

 Initiation

In transcription, one strand of DNA, the non-coding strand, is used as a template for RNA synthesis. As transcription proceeds in the 5' → 3' direction, and uses base pairing complimentarity with the DNA template to specify the correct copying, the DNA template strand is that oriented in the 3' → 5' direction. The strand that is not used as the template is called the coding strand, and has the DNA sequence that reflects that of the RNA produced.

Transcription begins with the binding of RNA polymerase to the promoter in DNA. In prokaryotes, the RNA polymerase is a core enzyme consisting of five subunits: 2 α subunits, 1 β subunit, 1 β' subunit, and 1 ω subunit. At the start of initiation, the core enzyme is associated with a sigma factor (number 70) that aids in finding the appropriate -35 and -10 basepairs downstream of promoter sequences. Transcription initiation is far more complex in eukaryotes, the main difference being that eukaryotic polymerases do not recognize directly their core promoter sequences.

Unlike DNA replication, transcription does not need a primer to start. The DNA unwinds and produces a small open complex and synthesis begins on only the template strand.

Elongation

Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases, so many mRNA molecules can be produced from a single copy of the gene. This step also involves a proofreading mechanism that can replace an incorrectly added RNA molecule.

Termination

Bacteria use two different strategies for transcription termination: in Rho-independent transcription termination, RNA transcription stops when the newly synthesized RNA molecule forms a hairpin loop, followed by a run of Us, which makes it detach from the DNA template. In the "Rho-dependent" type of termination, a protein factor called "Rho" destabilizes the interaction between the template and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex. Transcription termination in eukaryotes is less well understood. It involves cleavage of the nascent transcript, followed by template-independent addition of As at its new 3' end, in a process called polyadenylation.

Prokaryotic vs. eukaryotic transcription

• Prokaryotic transcription occurs in the cytoplasm alongside translation.
• Eukaryotic transcription is primarily localized to the nucleus, where it is separated from the cytoplasm (where translation occurs) by the nuclear membrane.

Measuring and detecting transcription

Transcription can be measured and detected in a variety of ways:

• Northern blot
• RNase protection assay
• RT-PCR
• In vitro transcription
• In situ hybridization
• DNA microarrays

Transcription factories

Active transcription units are clustered in the nucleus, in discrete sites called ‘transcription factories’. Such sites could be visualized after allowing engaged polymerases to extend their transcripts in tagged precursors (Br-UTP or Br-U), and immuno-labeling the tagged nascent RNA. Transcription factories can also be localized using fluorescence in situ hybridization, or marked by antibodies directed against polymerases. There are ~10,000 factories in the nucleoplasm of a HeLa cell, among which are ~8,000 polymerase II factories and ~2,000 polymerase III factories. Each polymerase II factory contains ~8 polymerases. As most active transcription units are associated with only one polymerase, each factory will be associated with ~8 different transcription units. These units might be associated through promoters and/or enhancers, with loops forming a ‘cloud’ around the factory.

Transcription initiation complex

Transcription factors mediate the binding of RNA polymerase and the initiation of transcription. The RNA polymerase only binds to the promoter after certain transcription factors are assembled. The completed assembly of transcription factors and RNA polymerase bound to the promoter is called the transcription initiation complex.

History

A molecule which allows the genetic material to be realized as a protein was first hypothesized by Jacob and Monod. RNA synthesis by RNA polymerase was 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.

Recently, Roger D. Kornberg won the 2006 Nobel Prize in Chemistry "for his studies of the molecular basis of eukaryotic transcription".^[2]

Reverse transcription

Scheme of reverse transcription

Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA in order to see a cell's genome. The main enzyme responsible for this type of transcription is called reverse transcriptase. In the case of HIV, reverse transcriptase is responsible for synthesising a complementary DNA strand (cDNA) to the viral RNA genome. An associated enzyme, ribonuclease H, digests the RNA strand and reverse transcriptase synthesises a complementary strand of DNA to form a double helix DNA structure. This cDNA is integrated into the host cell's genome via another enzyme (integrase) causing the host cell to generate viral proteins which reassemble into new viral particles. Subsequently, the host cell undergoes programmed cell death (apoptosis).

References

1. ^ Berg J, Tymoczko JL, Stryer L (2006). Biochemistry, 6th ed., San Francisco: W. H. Freeman. ISBN 0716787245. 
2. ^ Chemistry 2006. Nobel Foundation. Retrieved on 2007-03-29.

See also

• Genetics
• Molecular biology
• Translation - process of making RNA into polypeptides.
• Splicing - process of removing introns from RNA to make mature RNA (mRNA)
• Reverse transcription - process viruses use to make DNA from RNA
• Crick's central dogma - DNA is transcribed to RNA which is translated to polypeptides, never the other way around.

Further reading

• Lehninger Principles of Biochemistry, 4th edition, David L. Nelson & Michael M. Cox
• Principles of Nuclear Structure and Function, Peter R. Cook
• Essential Genetics, Peter J. Russell

External links

At Wikiversity, you can learn about:

Transcription (genetics)

• Interactive Java simulation of transcription initiation. From Center for Models of Life at the Niels Bohr Institute.