Transcription: A Starting Point

Transcription in Brief
Transcription is when the DNA strand is copied or transcribed into the four-base language of RNA.

This may seem a trivial statement, however when things can get complicated, it is an important statement to return to, as it provides the bigger picture of the significance of what is going on in the cell. (or test tube if you go all in vitro and such)

However, the details about transcription are anything but brief. For this post I will be covering the basic outline of transcription and from there, future posts will probably involve more precise, involved descriptions about the process.

Overview of Transcription

So, let's return to the DNA, sitting in its double helix form in the nucleus. During transcription one of its strands will act as a template, and will be denatured locally to allow access for an incoming ribonucleotide triphosphates (rNTPs). These rNTPs can be thought of as the building blocks of RNA, and bind to the complementary DNA bases with Watson-Crick base pairing. Remember however, that RNA uses uracil instead of thymine. RNA polymerase catalyzes a polymerase reaction to join the rNTPs from 5' to 3'. This is opposite in directionality of the template DNA strand, which goes from 3' to 5'. The sequence the RNA is transcribing is then the same as the nontemplate DNA strand.

What is polymerization? 

The mechanism works via a nucleophilic attack, where a nucleophile is a species that can donate an electron pair. In this case, the nucleophile is the 3' oxygen of the growing RNA chain, and it attacks the alpha phosphate, which is the first phosphate attached to the ribose of a chain of three of the incoming nucleotide that will be the next in the RNA sequence. This results in the formation of a phosphodiester bond, and the release of a phosphate (PPi). Again, the rNTPs are added from 5' to 3'.

Why is polymerization favoured? 

The high-energy bond between the alpha and beta phosphates of the incoming nucleotide (the first phosphate attached to the ribose, and the one next door) is replaced by a lower-energy phosphodiester bond between nucleotides. Also, another enzyme called pyrophosphatase, catalyzes cleavage of the released PPi into two moleules of inorganic phosphate, and pushes the equilibrium to favour chain elongation. In other words, the pyrophosphatase makes it easier for the phosphate to leave the high energy bond.

Some Conventions When Discussing Transcription

The site on the DNA where the RNA polymerase begins transcription is labelled +1, called the start site.

Downstream = the direction of transcription (+ from the start site)

Upstream = the direction opposite transcription (- from the start site)

Stages in Transcription 

Initiation 

1) Polymerase binds to promoter sequence in the duplex DNA with the aid of several general transcription factors (proteins). This is a "Closed Complex" as the DNA is still double stranded.

2) Polymerase melts duplex DNA near transcription start site for 12-14 base pairs, forming a transcription bubble, which seperates the DNA strands to allow rNTPs access to the DNA template strand sequence. It also allows the template strand to have access to the active site of polymerase so that it can catalyzes phosphodiester linkage. Now the DNA is called "Open Complex."

3) Polymerase catalyzes phosphodiester linnkage of two initial rNTPs.

Elongation

4) To undertake the elongation journey, polymerase dissociates from the promoter sequence and general transcription factors. Polymerase advances 3' to 5' down template strand, melting duplex DNA and adding RNTPs to growing RNA, while guiding the DNA strands so they hybridize at the upstream end of the transcription bubble.  The transcription bubble has a melted region of ~14 base pairs of which ~8 nucleotides at the 3' end of the growing RNA strand remain base-paired to the template DNA strand. The elongation complex consists of RNA polymerase, template DNA, and growing/nascent RNA. The speed of elongation is around 1000 nucleotides a minute.

Termination

5) At transcription stop site, polymerase releases completed RNA and disassociates from DNA. Once the polymerase is dissociated it is good to go find more DNAs and transcribe some more. It is not specific to a certain gene.

Structure of RNA Polymerases 

Structure of RNA polymerases is conserved between bacteria, archaea, and eukaryotic cells.

Bacteria RNA polymerase: 2 related large subunits (B and B'), 2 smaller identical subunits (a), and 1 copy of 5th subunit (w)

The w subunit stabilizes the molecule, no aid to transcription.

DNA bends sharply when entering the RNA Polymerase molecule.