Protein synthesis

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Protein synthesis is the creation of proteins via transcription and then translation on a ribosome, involving RNA polymerase, primersmRNA, 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.

Contents

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 [1]. 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.

Inhibition and Activation

Inhibition

A good example of inhibition and expression is the lac operon. The transcription of the lac operon, which is a polycistronic gene coding for B-Galactosidase, a permease and an acetylase,is regulated by the lac operator which is located upstream of the sigma factor binding site. In contrast activation of the operon is affected by CAP and binding to the CAP binding site which is downstream of the sigma factor binding site.

The repressor site is bound to by the lac repressor that acts as a brake to the RNA polymerase, it only works when there is a strong promoter for the sigma factor due to that if the brake is removed with a weak promoter transcription will not occur. the lac repressor is a product of the lacI gene made of 360 amino acids that forms a homotetramer. In the presence of Allolactose the lac repressor is induced it makes the repressor dissociate from the DNA so that transcription can continue.

Activation

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

Components 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[2]. The mRNA strand is attached to the tRNA strand by Hydrogen bonds and also attached to the ribosome.

The Mechanism

Initiation

The 30s sub unit of a Ribosome searches along the mRNA strand untill reaching the Shine Dalgarno sequence which is also known as the ribosome binding site this then goes along to find the first start codon AUG. In short the initiator charged tRNA (fmet-tRNAfmet) bind to the start codon and the 50s sub unit complexes with the 30s to form the 70s initiation complex. There are 3 sites in the ribosome A, P and E; A is holding the next tRNA with the corresponding amino acid, P is the tRNA molecule attached to which is the amino acid chain and in E is the site where there is an uncharged amino acid. the ribosome moves along as more amino acids are made in the elongation stage.

There are a number of factors involved in the those initial steps, these are IF1, IF2, IF3 and GTP.

Factors IFand IF3 bind to the 30s sub unit, IF2 complexes with GTP and binds with the 30s subunit as well. When the ribosome is attached to the mRNA and the first charged tRNA molecule is added IF3 is released. Then the binding of the 50s subunit causes IF2, IF1 along with GDP and Pi to be released giving energy. The complex can then go on to elongate the chain.

Elongation

To summarise this step, the charged tRNA molecule in the P site is holding the first amino acid or a chain of them. The next charged tRNA molecule binds to the next codon on mRNA in the A site. A peptide bond is formed between the amino acid in the P site and the one in the A site causing a release of the amino acid from the tRNA in the P site. the Ribosome moves up 3 base pairs and gets ready for the next charged molecule of tRNA. The uncharged tRNA in the E site is eventually released.

As with initiation there are a number of factors involved that drive the reactions. These are EF-Tu, EF-Ts, EF-G and GTP providing the energy. For the binding of the tRNA molecule and the creation of the peptide bond EF-tu, EF-Ts and GTP is needed and also an enzyme. For the moving of the Ribosome EF-G and GTP is needed.

Peptide Bond Formation

The peptide bond formation is catalysed by peptidyl transferase reaction this comes from the 23S rRNA.

Termination

Proteins that are named release factors recognise the stop codon on the mRNA chain. These factors are RF1, RF2and RF3. RF1 recognises UAA and UAG, RF2 recognises UAA and UGA, RF3 on the other hand is different it doesnt recognise these as such, it aids the other factors in completeing their termination. Polypeptide is then transferred to water, the uncharged tRNA molecule is released and the Ribosome detaches from the mRNA. Then the mRNA is degraded eventually.

References

  1. Hartl and Jones, Genetics - Analysis of Genes and Genome, p404-447
  2. http://www.uic.edu/classes/bios/bios100/lecturesf04am/lect14.htm
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