Transcription

Transcription is the process by which double-stranded DNA is made into single-stranded mRNA. This process is highly regulated and controlled to ensure the right amount of a specific gene is coded for at a specific time and place. It is said to be the first initial step in the process of gene expression in living organisms. Which is then followed by the process of translation.

mRNA

Proteins are synthesised in the cytosol, however, DNA does not leave the nucleus, therefore a copy of the gene coding for the desired protein is sent as a messenger to the cytosol from the nucleus. This is called messenger RNA 'mRNA', which is a single stranded molecule that is a complementary copy of the DNA strand it was synthesised from. RNA is made from ribose nucleotides that are free in the nucleus (rATP, rGTP, rCTP and rUTP).
Pre mRNA is first transcribed which contains non coding Introns these introns are spliced out to leave mRNA that contains only the coding exons.

mRNA contains specific sequences which code for different amino acids. In transcription of proteins there are certain sequences which code for start and stop signals for protien synthesis. Generally, Methionine is a start codon. Methionine only has one series of bases coding for it (AUG) compared to Serine and Arginine which have 6 sets of coding mRNA each. There are three stop codons which code for the end of a protein molecule (UAA, UAG, UGG). 

Code for all 20 amino acids and stop codons:

	Second Position
First Position (5' End)	U	C	A	G	Third Position (3' End)
U	Phe Phe Leu Leu	Ser Ser Ser Ser	Tyr Tyr Stop Stop	Cys Cys Stop Trp	U C A G
C	Leu Leu Leu Leu	Pro Pro Pro Pro	His His Gln Gln	Arg Arg Arg Arg	U C A G
A	Ile Ile Ile  Met	Thr Thr Thr Thr	Asn Asn Lys Lys	Ser Ser Arg Arg	U C A G
G	Val Val Val Val	Ala Ala Ala Ala	Asp Asp Glu Glu	Gly Gly Gly  Gly	U C A G

Double stranded DNA

DNA is double stranded as opposed to mRNA which is single stranded, therefore only one strand of DNA is copied. The copied strand is called the 'template strand' (also known as the 'sense strand'), the other strand is called the 'non-template strand' or the 'coding strand' (also known as the 'anti-sense strand') and will have the same base sequence as the RNA strand produced. mRNA is synthesised by the enzyme 'RNA polymerase', however, in order for the RNA to synthesise mRNA it must bind to a single strand of DNA. The DNA must be unwound and unzipped, which is done via an enzyme called 'DNA helicase', which unwinds and unzipps the double stranded DNA at the loci of the gene to be transcribed, causing an area of single stranded DNA to be accessible to the RNA polymerase. This unwound section is known as the transcription bubble.

Unlike DNA polymerase which requires an RNA primer to iniate replication, RNA polymerase does not.

Promoter regions

RNA polymerase must recognise and bind to a region upstream of the gene being transcribed called the 'promoter region'. This region is a sequnce of bases that determines the strength of the binding of RNA polymerase, to the DNA strand and therefore determining the efficiency of trancription of the gene it is accossiated with. If the promoter is a strong promoter, then RNA polymerase binds strongly to the DNA strand. If the promoter is a weak promoter, then the RNA polymerase can become hindered and can even unbind from the DNA strand. The promoter region strength is determined by how promoter sequence compares to other promoters on separate genes. When different promoters are compared, a sequence of bases can be determined that are most common in all the promoter sequences of that type, this is called a 'consensus sequence'. The closer the promoter sequence is to the consensus sequence, the stronger the promoter and the stronger the binding of the RNA polymerase.

Sigma factors and Eukaryotic Transciption factors

RNA polymerase cannot bind to the promoter region unless a sigma factor is present. Sigma factors ensure that the RNA polymerase binds to the correct promoter region, this is another method in which transcription is regulated. The sigma factor binds to the RNA polymerase via specific binding sites on its structure and forms a ‘holoenzyme’, i.e. sigma factor + RNA polymerase = holoenzyme.

In E. coli the holoenzyme recognises specific Consensus sequence at -35 and -10 within the promotor region. At the -35 sequence the DNA remains double stranded, and a closed complex is formed, however at the -10 sequence (or Pribnow box) about 14 bases are melted, and the closed complex becomes a Transcription_Bubble with exposed bases.

Unlike bacteria, Eukaryotic cells has mor ethan one transcription factor, these transcripsion factors are: TF2D, TF2A, TF2B, TF2F, TF2E, and TF2H, binding in that order onto the TATA box. They have diffenrt fucntions during the intiation process.

Initiation

Once the sigma factor has bound to the RNA polymerase, the RNA can bind to the promoter region upstream of the gene on the single stranded DNA. The RNA is then free to transcribe the gene. Free ribose nucleotides bind to the DNA sequence via complementary base pairing. Instead of the base Thymine found in DNA, the base uracil is used in RNA. The RNA polymerase joins the nucleotides together via strong covalent phosphodiester bonds, this forms the single strand of mRNA. This process is called initiation.

In Eukaryotic cells the initatition starts with TFIIH binding to the RNA pol II and phosphorylating the N terminus of the polymerease. When the double stand is broken the RNA starts to bind to DNA at teh start site.

Elongation

When 10 nucleotides of mRNA have been synthesised, the sigma factor is released from the RNA polymerase. The RNA polymerase continues to transcribe the gene. This is called elongation, where the RNA polymerase moves along the DNA strand and creates a single strand of mRNA that is complimentary to the DNA sequence. Only 8 nucleotides of mRNA remain attached to the DNA sequence at a time. The mRNA peels of the DNA sequence but still remains attached to the rest of the mRNA molecule. Once the mRNA has been synthesised from specific nucleotides, an enzyme recombines the two DNA strands and rewinds it into its helix structure. This occurs while the RNA polymerase is still transcribing during elongation.

Termination

Once the gene has been synthesised, the RNA polymerase must stop transcribing or it would continue to transcribe uncontrollably. The sequence present at the end of a gene sequence that stops transcription is called the ‘terminator sequence’.

In eukaryotes, the termination occurs when the RNA polymerase complex reaches a chain termination sequence, usually consisting of bases TTATTT (or AAUAAA when transcibed onto the mRNA). Termination of the transciption occurs abouut 10-35 bases downstream. The RNA is cut from the DNA by endonuclease. 

In prokaryotes, such as E.coli, however, there are two types of terminator. The first is called ‘Factor independent termination’. The sequence of bases at the end of the gene have a region rich and G+C bases with a sequence in-between, followed by 4 to 10 A+T bases. Once this area of the gene has been transcribed, the section of rich G+C on the mRNA molecule bind together by complimentary base pairing. This forms a hairpin structure at the end of the mRNA molecule. This hairpin structure has properties that cause the RNA polymerase to pause in transcribing the gene. Once paused, the RNA polymerase unbinds from the DNA molecule and releases the complete mRNA molecule, thus terminating transcription. There is also a second method of termination. This is called Rho dependant termination. This involves a helicase enzyme called a Rho factor, which unwinds the mRNA from the DNA molecule faster than it does naturally. The Rho factor unwinds the mRNA until it reaches the RNA polymerase. This causes the RNA polymerase to pause and stop transcribing proteins, causing the RNA polymerase to unbind from the DNA and the complete mRNA molecule to be released.

The mRNA molecule then exits the nucleus into the cytosol, where it will be translated into proteins that the cell requires for the second step of gene expression known as ‘translation’.