Transcription

Transcription is the process by which small double-stranded DNA, coding for a single or handful of genes, is used as a template to make single-stranded mRNA. The complementary RNA strand is named the gene transcript. 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 initial step in the process of gene expression in living organisms and involves the polymerisation of ribonucleotides, creating a phosphodiester bond between a 3’ OH and a 5’P.

Transcription can occur in both prokaryotes and eukaryotes. In eukaroyes, transcription occurs in the nucleus. It produces an mRNA strand complementary and antiparallel to the template DNA strand which also has an identical base sequence and direction to the non-template strand, however with the substitution of Uracil for Thymine. This is then followed by the process of translation.

mRNA

Proteins are synthesised in the cytosol (translation). However, as DNA does not leave the nucleus, 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 antisense DNA strand it was synthesised from. RNA is made from ribose nucleotidesthat are free in the nucleus (rATP, rGTP, rCTP and rUTP).

RNA becomes a functional mRNA molecule, that can leave the nucleus when the 5’ and 3’ ends have been modified by splicing. Pre-mRNA is first transcribed which contains non-coding Introns. These introns are spliced out via a ribozyme known as the splicesome, to leave mRNA that contains only the coding exons.

mRNA contains specific sequences called codons, 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 compared to mRNA which is single-stranded, hence 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 refered to as 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 unzips 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 initiate replication, RNA polymerase does not. RNA polymerase recognises and binds to a promoter region in order to start the process of transcription.

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 sequence of bases that determine the strength of the binding of RNA polymerase, to the DNA strand and therefore determining the efficiency of transcription of the gene it is associated 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 Transcription 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 RNA polymerase holoenzyme in prokaryotes contains the specific Sigma factor: σ70. 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.

Eukaryotic cells differ from bacteria cells in transcription as they utilise multiple transcription factors to recruit RNA polymerase. The type of RNA polymerase which is recruited depends on which type of RNA is being transcribed; RNA pol I, II and III transcribe rRNA, mRNA, tRNA respectively. Collectively the RNA pol and transcription factors form the Pre Initiation Complex (PIC). For example to transcribe mRNA, transcription factors: TFIID, TFIIA, TFIIB, TFIIF, TFIIE and TFIIH, which usually bind in that order, along with the RNA polymerase II onto the TATA box (if it is present). The TATA box is a consensus sequence, which means it is a sequence of DNA which has a similar function/structure in various other organisms. It is found in the eukaryotic promoter in the basal region.

The transcription factors have different functions during the initiation 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. Promoters only exist just upstream of genes, ensuring it is always genes that are translated and not random bits of non-coding 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, there are 4 potential elements that the core promoter region may contain but it is unusual that the promoter region will have all four. The four elements are the TATA box found at -31 to -26, the initiator site at -2 to 4, downstream promoter element at +30 and finally the B recognition element at -37 to -32. The numeric values are in retrospect to +1 being the transcription start site.In Eukaryotic cells the initiation starts with TFIIH binding to the RNA poymerase II. TFIIH has helicase activity which drives promoter melting over the transcription start site which separates the template strand. This requires ATP, unlike promoter melting in Prokaryotes. As the DNA is open, this is now the open complex.

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, RNA polymerase dissociated from the DNA and an enzyme recombines the two DNA strands and rewinds it into its helix structure, meaning that only 12-17 DNA bases are exposed at any one time. This occurs while the RNA polymerase is still transcribing during elongation. RNA binding proteins bind to single-stranded RNA nucleotides to stabilise it, and to prevent any unwanted reactions or binding with other proteins.

Termination

Once the gene has been synthesised the RNA polymerase must stop transcribing or it will uncontrollably continue to keep transcribing. 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 transcribed onto the mRNA). Termination of the transcription occurs about 10-35 bases downstream. The RNA is cut from the DNA by endonuclease.

In prokaryotes, such as E.coli, there are two types of terminator. The first type being ‘Factor independent termination’, the sequence of bases at the end of the gene have a region rich in G+C bases with a sequence in between, followed by 4 to 10 A+T bases. Once the region has been transcribed, the section with rich G+C on the mRNA molecule bind together by complementary base pairing. This forms a hairpin-like 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-dependent 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.

Eukaryotic pre-mRNA requires extensive modification. A 7-methylguanosine cap is added to the 5’ end, and a poly-A-tail is added onto the 3’ end. The significance of these modifications is the prevention of mRNA degradation, and also to promote its binding to ribosomes, increasing the translational efficiency of mRNA. Another necessary modification is the removal of introns by the spliceosome. Modification is a crucial aspect of unlocking the information from the genetic code as otherwise the entire reading frame is altered, which would translate into a dysfunctional protein.

The mRNA molecule then exits the nucleus via a nuclear pore, into the cytosol, where it will bind to a ribosome and be translated into the proteins that the cell requires for the second step of gene expression known as ‘translation’.