(Transcribed from Dr. Cadilla’s lecture, 31 Mar 2000 by Brian Buschman)
Return to Semester One Goodies
Return to The Unofficial Ross Page
The mRNA is read in the 5’ to 3’ direction in blocks of three bases called codons. It begins at the start codon, AUG, and ends at one of the three stop codons. Transcription is carried out by ribosomes. In the prokaryote ribosomes have the 30s and the 50s subunits. In eukaryotes it is the 40s and 60s subunit. When the two subunits are assembles the ribosome is said to be 70s in the prokaryote and 80s in eukaryotes. Note that s is a unit related to the sedimentation factor from the centrifugation of cytoplasm which is why you cannon sum the two subunit sizes.
Ribosomes have two sites called the A-site (aminoacetyl tRNA site) and the P-site (peptidyl tRNA site).
tRNA is the “adapter” molecule between the codon on the mRNA and the amino acid residue. They have a region that is complimentary to the codon known as the anticodon. There is some level of variation in the base pairing between the anticodon and the codon known at wobble. It says that for some bases in the anticodon more then one bases in the codon is able to pair. The combinations include:
b
|
This one can pair with |
Any of these |
|
C |
G or I |
|
A |
U or I |
|
G |
C or U |
|
U |
A, G or I |
|
I |
C, U or A |
tRNAs are prepared for translation by aminoacetyl tRNA synthetase which is an enzyme that attaches the amino acid to the rRNA. In the process of binding the amino acid to the tRNA they convert one ATP to AMP and pyrophosphate.
Protein synthesis begins with the initiation factors that guide the smaller subunit with a bound N-formylmethionine to bind to the AUG sequence on the mRNA. In eukaryotes they are able to recognize the proper AUG sequence. Prokaryotes have the Shine-Dalgarno sequence located nearby and upstream of the proper AUG at which they need to start at. In prokaryotes it can bind to any AUG sequence but in eukaryotes it is able to determine which is the proper AUG sequence for it to start at. As you can probably assume N-formylmethionine will be the first base to be added to any transcribed peptide.
The initiation factors are released and the larger of the subunits (50s or 60s) will bind to the 30s or 40s subunit with the aminoacetyl tRNA with formylmethionine in the P-site. Then the aminoacetyl tRNA of the next codon will bind to the A-site. The peptide bond will then be formed between the two and what is in the A-site will be translocated to the P-site and what is in the P-site will be ejected. The process repeats like that until a stop codon is encountered. In this case a release factor binds in the place of an aminoacetyl tRNA and the ribosomes are then removed from the mRNA.
Most of the energy used in translation comes from GTP rather then ATP. The energy requirements are as follows:
As you can see form this count it takes four high energy bonds for the addition of each amino acid to the chain.
The direction of transcription is 5’ to 3’ along the mRNA. It is possible to have any number of ribosomes working on the same mRNA at the same time. This combination of an mRNA and multiple ribosomes is called a polysomes.
Polypeptide synthesis is not the last step in protein formation. The protein still needs to be folded into it’s higher order configuration, which may happen spontaneously or require the help of chaperones. The protein must also be transported to it’s proper destination.
There are a number of post-translational processes that may happen that include:
1) Removal of the signal sequence.
2) Disulfide bond formation.
3) Glycosylation.
4) Amino acid modifications.
5) Partial proteolytic cleavage.
There are two types of ribosomes, free and membrane bound. The free ribosomes produce cytoplasmic peptides and ER bound ribosomes produce membrane bound and secretory proteins.
They become ER bound partially through translocation. Initially free ribosomes translate the signal sequence which will bind to a signal recognition particle (SRP). The SRP migrates to the ER and binds to the SRP receptor. Translocation then continues into the ER.
Oligosaccharides can be either linked to the –OH group of serine or threonine or to the amide group of asparagines. If linked to the –OH group they are O-linked oligosaccharides that are formed by the stepwise addition of monosaccharides in the golgi. The steps are catalyzed by glycosyl transferase. N-linked oligosaccharides are formed in the ER by the addition of sugars to the amide group in asparagines. In this case the oligosaccharides is formed and then transferred to the peptide as a large unit.
Lysosomes are synthesized from the secretory pathway but their enzymes are marked by a mannose-6-phosphate to identify that they belong inside the lysosome. In the case of I-cell disease the mannose-6-phosphate is missing and these enzymes are secreted by exocytosis which causes all sorts of problems.
Return to Semester One Goodies
Return to The Unofficial Ross Page