Protein synthesis

Protein synthesis is the creation of proteins via transcription and then translation on a ribosome, involving RNA polymerase, primers,  mRNA, tRNA and rRNA. It occurs in both Eukaryotic and Prokaryotic cells although there are certain differences like splicing occurs in different places and of course there isn't a nucleus in prokaryotes so no movement between membranes is involved to get the mRNA strand out of the cell.

Transcription

Transcription is the copying of DNA in the nucleus into pre-mRNA. For a gene to be synthesised into a protein it needs certain pathways within cells to occur which causes binding of transcription factors to the promoter sequence on DNA. There are 3 parts to transcription, chain initiation, chain elongation and chain termination the chain initiation and termination are detailed below due to it being different in different types of organism.

Chain Elongation

Componants

RNA polymerase - Enzyme that catalyses the making of covalent bonds down the sugar phosphate backbone of an mRNA strand. Also is dependant on have magnesium ions present for the process to work.

rATP, rGTP, rCTP, rTTP - these are normal nucleotides with a extra 2 phosphate groups.

Mechanism

The mechanism for this is that incoming rNTP's attach to their complementary Base. The rNTP's are basically free living nucleotides each with 3 phosphate groups attached instead of one and an alpha, beta and a gamma. The alpha phosphate is attached to the ribose sugar which in turn is then attached to the base. As the Polymerase works down the chain it breaks the bond between the alpha and the beta phosphate. Then creating a bond between the currect nucleotide and the previous nucleotide. the polymerase then moves up to join the next rNTP.

Chain Initiation and Termination

Prokaryotes

In Prokaryotes initiation of transcription is different from that of Eukaryotes, at -10 and -35 these sequences are located upstream of the start site of transcription. There are specific sequences at each site that enable a Sigma factor to bind. At -10 there is a sequence similar to TATAAT, the Pribnow box, and then at -35 it is similar to TTGACA . The sequences given previously are the perfect sequences and so if they are present the Sigma factor would bind very tightly to the DNA strand. How tightly the Sigma binds to the DNA determines the amout of expression of that gene and so is a regulatory effect related to how many is needed. This Sigma factor then in turn binds to the RNA polymerase forming a holoenzyme, this now allows transcription to begin.

When the RNA polymerase reaches the termination sequence the polymerase is distrubted by a hair pin sequence formed by the transcribed RNA strand folding back on itself and forming hydrogen bonds. This hairpin weaken the hold of the RNA strand with the DNA by looping the mRNA strand leavng only U's or T's attached and these having less hydrogen bonds than the other bases it can disjoin from the DNA causing the polymerase to stop working. 

The other difference in prokaryotes is that the arrangement of genes are ordered by function, so all the similar funtioning genes are kept in the same region. So instead of having multiple promoters and terminators, it normally has 1 per section of similar genes this is called polycistronic. But it also will cause problems if there is a mutation in the promoter region of the sequence as none of the genes related to each other will be transcribed, so a none functioning end product. This is due to prokaryotes being simple organisms.

Eukaryotes

In Eukaryotes the initiation of transcription normally occurs at the -25 TATA box sequence. This is initiated by the TFIID complex binding, the TFIID then binds other transcriptional factors that regulate the level of expression and also an RNA polymerase. once this complex is formed then transcription can begin. On termination of the transcription the terminator sequence is reached  (AAUAAA) and the polymerase finishes arouns 10-35 nucleotides past that sequence. unlike in RNA where the mRNA strand remains attached it is cut by a restriction enzyme allowing it to be spliced and then transported out of the nucleus.

Unlike in Prokaryotes DNA is not polycistronic it is monocistronic so there is a promoter for every gene and a terminator for every gene. This is due to the complexity of Eukaryotes but also that the arrangement of genes are not ordered on the DNA sequences. Also at the end of Eukaryotic transcription there is something called a cap added at the 5' prime end and a polyadenine tail added to the 3' prime end. The cap is created by the addition of a modified Guanosine, this is essential for the binding to a Ribosome in translation.

Splicing

Splicing occurs in the nucleus by the use of a spliceosome looping out the introns then cutting them out and binding the exons together leaving a strand of only exons. This is the pre-mRNA maturing and turning into mRNA to leave the cell.

Translation

Componants involved

mRNA

mRNA is a copy made of the DNA by RNA polymerase II and spliced to take out all the introns. This is a single polynucleotide strand that have codons made up of Adenine, Guanine, Cytosine and Uracil and instead of a deoxyribose in the sugar phospate backbone there is a ribose molecule. 

tRNA

This is a single polynucleotide strand that folds back on itself to form hydrogen bond in the shape of a clover leaf. This molecule on one end has an anticodon which is complimentary to the codon on the the mRNA strand which it attaches to. On the other end there is a specific protein that is able to detach and form part of a polypeptide chain.it is about 70-90 nucleotides long and contains varient and semi-varient regions. it also contains modified bases such as iosine.

Ribosome

The Ribosome is the location of the translation of proteins, the Ribosome has 3 tRNA binding sites the P site which holds the tRNA molecule with the polypeptide strand, the A site which binds to the tRNA molecule with the next amino acid to by hydrolysed and the E site which holds the tRNA molecule to be discharged. The mRNA strand is attached to the tRNA strand by Hydrogen bonds and also attached to the ribosome.

The Mechanism

Initiation

The small subunit of the Ribosome