Laith AL-Mustafa Protein synthesis Nabil Bashir 10\28\2015 http://1drv.ms/1gigdnv 01 First 0
Protein synthesis In previous lectures we started talking about DNA Replication (DNA synthesis) and we covered the first part of gene expression, which is: 1- Transcription: RNA synthesis In this lecture we will talk about the other part of gene expression, which is: 2- Translation: protein synthesis SO, Let's learn more details about translation (protein synthesis) Ribosome s structure It s a machine for protein synthesis (translation), and it s simply composed of protein and rrna (like chromosome and how it is composed of protein "histone" and DNA). In this figure we see a polypeptide (protein) that is undergoing protein synthesis and an mrna that is being used as a template in protein synthesis. 1
We see Ribosomal protein subunits: Eukaryotic system Prokaryotic system Large subunit 60S 50S Small subunit 40S 30S Assembled subunits including small and large proteins and RNA molecules 80S 70S What does S stand for? Svedberg unit is sedimentation (precipitation) coefficient expresses the rate at which the molecule move in a centrifugal field It is determined by the size, shape, and density of the molecule So... here, in this table we can see that assembled forms will not have the summation of two sizes so 50S + 30S in Prokaryotic system will not give you 80S assembled ribosome but it will give 70S, the same thing in Eukaryotic system it will give 80S not 100S Again why does this occur? Because this is a sedimentation (precipitation) unit, so when things are sediment they depend on their density and shape; in this case when two subunits are assembled they produce different shape with different density that will sediment at 70S NOT 80S. 2
Finally, we see (E, P, and A) on the figure, each letter represent a site in the small ribosomal subunit either in Prokaryotic or Eukaryotic system and each has a function in protein synthesis. We will talk it in the next lecture. Basics of protein synthesis: mrna is decoded or read in the 5' -to- 3' direction, one codon at a time and the corresponding protein is synthesized in the amino-to-carboxyl direction (N-terminal to C-terminal) by sequential addition of amino acids to the carboxyl end of the growing peptide chain The amino acids arrive at a growing chain in activated form as aminoacyl-trnas (charged trna), created by joining the carboxyl group of an amino acid to the 3' end of a trna molecule this linking of amino acid to its corresponding trna is catalyzed by an aminoacyl-trna synthetase 3
It s clear here trna and amino acids (we don t see mrna) and how these charged amino acids bind to trna they are leaving the trna and incorporated once at a time sequentially this is what happens during peptide bond formation between amino acids, these reaction will take place in ribosomes and the (E P A) sites will participate in this process Fidelity of translation Fidelity: the degree of exactness when the gene replicate, transcript and translate. DNA Replication DNA Transcription RNA Translation Frequency Of mistakes 1\1000000 (10^-6) 1\1000 (10^-3) 1\100000 (10^-5) For the proteins That composed of 1000 amino acids Example: if you want to synthesis a protein from a gene and that protein composed of 1000 4
amino acids the probability of incorporation wrong amino acids is (10^-5) The genetic code: -Don't memorize this table. BUT it has some features: Redundancy: That means one amino acid could have more than one codon [we have 20 amino acid, it is supposedly to have 20 codon. However, we have 64 codons] Q: Where are the codons of the modified amino acids? It has the same codon of its parent amino acide. In collagen we see hydroxyproline, hydroxylysine and gama carboxyglutamic acid these modified amino acids undergo posttranslational modification [hydroxylation, oxidation, carboxylation, methylation, acytlation] after translation of its parent amino acids. Not randomness: The first two nucleotides in the codon are the most important for amino acid and the third one is not important as the first and second. 5
For example: {GCA} {GCU} {GCC} {GCG} These four codons have the same first two nucleotide {GC} but differ in the third nucleotide, but all of them code the Alanine amino acid this role is found in most of the amino acids. Wobble: The 3 rd could be anything or biased towards purines or pyrimidines, For example: Histamine has only two codons {CAU} {CAC}, note that the third nucleotide is biased toward pyrimidine (U or C). Punctuation features: It means that translation starts from a specific point and end at specific point. All peptide chain start with methionine because the start point in translation is the methionine codon (AUG), also known as initiator codon. If you go to a protein bank and look for the N-terminals of different proteins you will not see methionine, because they are processed after folded by chaperone. Also there are three stop codons to terminate translation. Once the ribosome reaches any stop codon it will stop translation, these the three stop codons are {UAA} {UAG} {UGA}. All these features are Universal, because every cell uses this system even in the prokaryotic organism. 6
Inosine It's deaminated adenosine or adenine found frequently in trna specifically in the anticodon, provides flexibility for each trna to base pair with the different codons on mrna.so only one trna can use for different codons of the same amino acid We said that the third nucleotide in the codon could be any of the four nucleotides, so if the first nucleotide in the anticodon is inosine it could base pair with most of other nucleotides in the third position of the codon, this is the flexibility that is offered by inosine to fit and adapt with the wobble system in the genetic code. It is highly economical for the cell in term of synthesis of different type of trna molecules -Don t memorize this just understand the idea 7
part of trna sequence first nucleotide in the anticodone part of mrna sequence third nucleotide in the codon maybe (C,U,G,A) This is the Codon/anticodon base pair and this is what we are going to see during protein synthesis or translation, this base pairing is antiparallel, since translation of mrna in ribosome begin from 5' to 3' so base pairing of anticodons in trna will be 3' to 5 because it is antiparallel. It is possible for amino acid to has more than one trna Activation of amino acids To use an amino acid in protein synthesis it must be activated to use as a monomer to make polypeptide polymer, this process is similar to use of uridine diphosphate glucose (UDPG), which is the activated form of glucose as a monomer in glycogen synthesis. Q: How are amino acids activated? Each amino acid has its own enzyme for activation and that enzyme is called aminoacyl-trna synthatase so at least we have 20 types of aminoacyl-trna synthatase. 8
This smart enzyme will grab specific amino acid with it's trna and put it in close proximity and proper orientation by define the anticodon of this trna, but at first it will activate the amino acid by ATP then link it to its proper trna, after that it will release it as activated aminoacyltrna molecule is ready for translation. Q: Why aminoacyl-trna molecule highly reactive? This molecule has an acyl group carrying more energy and this energy will be used in peptide bond formation. This group will be attached to the 3' prime hydroxyl group of adenine amino acid at the 3'end of trna {CCA end} This acyl group has a very unstable bond and if exposed to water, it will be hydrolyzed so it must be covered and protected from water and we will see how it must be protected from water... later on. very unstable bond un (acyl group) 9
-This is the aminoacyl-trna synthetase, it has an editing site and an activation site and we see a specific trna that binds to this enzyme. Supposedly, if the wrong amino acid comes to the activation site or wrong trna not compatible with amino acid comes this enzyme, this editing site will discover this incompatibility and it will discard the wrong amino acid or trna. -So this enzyme is a very powerful enzyme in catalyze amino acid activation and editing any mistakes in trna and amino acid linking. This beautiful figure resembles the tertiary structure of aminoacyl-trna synthetase, in this figure we must note how the lower domain of this enzyme bind to trna and recognize it's anticodon, if it recognizes the anticodon properly it will bring the proper amino acid for linking. lower doman of enzyme 10
Recognition site on trna : these spots are the reading site where aminoacyl-trna synthetase read trna, not only the activation and editing site but the whole enzyme contribute in reading trna in order to be sure that the proper trna is linked. Ribosomal RNA : Component of ribosome, in prokaryote 16S rrna is found in 30S subunit but 23S and 5S rrna are found in 50S subunit, 23S rrna is responsible for catalyzing the formation of peptide bond during protein synthesis so it acts as peptidyl transferase (ribozyme) These lobes of this rrna are important for stabilization of the secondary structure that will give the tertiary structure of the protein (ribosomal subunit) 11
-In prokaryotes and eukaryotes all polypeptide chains must be start with methionine {AUG},But only in prokaryotes this methionine must be formylated (the formyl group must be added to this methionine) while it's hooked to initiator trna (called initiator if it link to methionine -After methionine link with its initiator trna by aminoacyl-trna synthetase this methionine will trans-formylated (adding formyl group to methionine) the source of this formyl group is N^10-formyl-tetrahydrofolate This formyl group is used to protect the acyl bond that link amino acid to trna initiator from water so it prevent water from touch this high energy unstable group. In eukaryotes there are initiator factor that protect methionine from water but not by formylation. 12
Prokaryotic translation initiation sequences purine-rich region {shine-dalgarno} sequence Shine Dalgarno sequences : purine-rich region in 5'end of mrna, is a ribosomal binding site in a prokaryotic mrna, recognized by a complimentary sequences found in the 16S rrna which is a component of the small ribosomal subunit this importance of shine Dalgarno sequences : It helps in grabbing and holding the mrna by a complementary base pairing with 16S rrna of the small ribosomal subunit because everything in prokaryotic cell found in the cytoplasm and this will tell the cell from where it will start translation. 13
the first {AUG} comes after that shine-dalgarno sequence will be the starting or the initiating codon to start the translation in prokaryotic system. Q: How bacterial cell determine the site of translation in specific mrna {translation in prokaryotic system}? after the ribosome with its16s rrna holding the mrna the first {AUG} after that point will be the initiator {AUG} and translation will start from that codon. 14