Translation (Protein synthesis)

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Central dogma of molecular biology describes gene expression from DNA to protein. The flow of information in the cell: DNA is transcribed into RNA which is translated to proteins. DNA -> RNA -> Proteins.
Translation is the synthesis of polypeptides. 3 phases (as most polymerization processes): initiation, elongation and termination; and ribosome recycling. In eukaryotes the biggest difference is in initiation. Its importance is reflected in biomedicine.
Molecules of DNA store genetic information and the order of nucleotides determines the information. There is a specific code - the genetic code to unlock the genetic information. ''Universal'' same code applies to virtually all organisms although exceptions to the genetic code and a theory of evolution of the code are contained in Ohama et. al.   search it - article 84/2/58 something
The genetic code is triplet: Codon = 3 nucleotides. 4 nucleotides code 20 amino acids required for life. 1:1 ratio isnt possible, nor 2:1 but 3:1 or 3nucleotide code gives 4x4x4 = 64 possibilities.

{{{ Small not important section It was shown in 1961 by Crick, Brenner and co and used suppressor mutations in T4 phage. The mutant phage only grows on 1 of 2 E.coli strains inducing a 2nd mutation could reverse effects of original mutation i.e. phage could grow on both strains like wild type (wt) called a revertant. Revertant isn't identical to wt T4. Consider the sentence: The fat cat ate the big rat. Delete c creates nonsense. The fat ata tet heb igr at. A second change could restore some original sense eg. Add A:    The fat ata ate the big rat => revertant but revertant isnt identical to original. Found that mutations adding or removing 3 nucleotides forms revertants -> codons in blocks of 3.  The fat cat ate the big rat. The cat ate the big rat. Advantages of a degenerate genetic code: mutations may be silent: do not change the amino acid coded for, hence mutations are less likely to be damaging, degeneracy provides some ''genetic stability''. Restricted supply of nucleotides, etc. may lead to the evolution of an organism with DNA/RNA of a different base composition while still coding for the same proteins. Reading frames. A gene will have a correct ''reading frame'' -> the right triplet sequence to be translated. Therefore there are 2 incorrect reading frames. Overlapping or non overlapping codes. Non overlapping each consecutive AA coded by consecutive codon. Overlapping each consecutive AA has 1 or 2 nucleotides in common with previous. Mutation of 1 nucleotide only affects 1 AA. Decoding triplets. Homopolymers - UUUUUU, AAAAAAA synthesized in vitro with polynucleotide phosphorylase no DNA template required. Triplet binding assay: synthesize known triplets. Mixed with  tRNA carrying labelled AA and ribosomes. Correct tRNA-AA, complex formed - retained on filter.    Repearing copolymers:   Synthesize long chain of repeating sequences. UGUGUG gives UGU (cysteine), GUG (valine), UUGUUG gives UUG (leucine), UGU, GUU (valine). Produces polypeptide of repeating AAs. Aminoacyl synthetase mechanism: enzyme activates AA by covalently binding with ATP between its 5'P and the -COOH of the A. Called aminoacyladenylic acid and remains as a complex with the specific synthetase. AA is then transferred to the 3' OH group on the ribose 3' end of one of the tRNAs for that AA. Specificity for AA: enzyme specificity for similar substrates is usually about 1:100. Miss-incorporation of amino acid is 1:10000. Proof-reading. Based on attachment and hydrolytic active sites}}}    

It is written in a linear sequence of nucleotides - ACGT. We 'read' the code from the gene's coding strand not the template strand of DNA. Transcribed to RNA: ACGT -> ACGU. Sequence of 3 nucleotides is a codon that specifies a given amino acid (AA). Collinear: order of codons same as AA order. 3 nucleotides - a triplet code. Triplet code is the most effective way of coding 20 AAs from 4 letters. In 18 of 20 AAs, more than one codon specifies a given AA (degenerate). The genetic code is degenerate. 4 nucleotides code 20 amino acids required for life - 3:1 or 3 nucleotide code gives 4x4x4=64possibilities. All amino acids except Methionine and Tryptophan have more than one codon. They show 2,3,4 and 6 fold degeneracy. Its largely associated with the 3rd position. Potential problem concerned with AA recruitment by tRNA and recognition of degenerate codons is explained by Cricks' wobble hypothesis, considered with role tRNA in lecture.
Proteins: 1 or more polypeptide chains. The polypeptide chain is a polymer of amino acids. AAs are the building blocks of proteins.Polypeptide chain
Translation: formation of polymer of amino acids in a given sequence called a polypeptide. It takes place on ribosomes using RNA as the template. It uses an adaptor called transfer RNA (tRNA) to direct correct a.a. to the codon. tRNA contains a complementary sequence to the codon called an anticodon. The RNA is both the main structural and functional component of ribosomes. The components of translation are mRNA, tRNA, ribosome (including rRNA).
tRNA - the adapter molecule. Francis Crick in 1958 suggested: an adapter molecule carries an amino acid to the  template (mRNA), the adapter fits on the mRNA itself, it would require 20 adapters - 1 for each AA and the adapter itself would contain nucleotides allowing base pairing as in double stranded DNA. Crick was largely correct. tRNA acts as an adapter! It is a special class of RNA. It is stable and in both eukaryotes and prokaryotes, contains 75-90 nucleotides. Each amino acid is attached to a specific tRNA. It is written as
tRNA<sup>ala

 It has virtually identical sequences between prokaryotes and eukaryotes for any given a.a.. The tRNA transports a,a. The tRNA transports AA to the ribosome. The ribosome attaches a.a. to growing polypeptide chain. tRNA (structure) is formed as larger precursor (e.g. tRNA^ala 126-77). Post transcriptional modification introduces unusual bases: Purines: insosine (I), 1 methyl inosine (mI), 1-methyl guanosine (mG), dimethylguanosine (m2G) and Pyrimidines: pseudouridine (ψ - psi), ribothymidine (T). Holley cloverleaf model - 2D model produced by base pairing. Loops contain unusual bases that do not form pairs. The anticodon loop is found in all tRNAs. With tRNA^ala the anticodon is 3'-CGI - 5'. Inosine (purine) can bind with A,C or U (wobble). All tRNAs have at 3' end CGA with adenosine covalently bound to amino acid. The 5' end is always G. Crick's wobble hypothesis and tRNA: anticodon base pairing with the third base in the codon may ''wobble'' (change its conformation). It can form additional (non Watson and Crick) base pairs and bind to more than one codon. One tRNA may accomodate up to 3-fold degeneracy. Anticodon 1st position - Codon 3rd position: C - G, A - U, U - A or G, G - C or U, I - U,C or A. 3 tRNAs for Serine (give example; slide 20 of 65). Charging tRNA: AAs are added to tRNAs (charging) by enzymes called aminoacyl tRNA synthetases. 20 AAs and 61 triplets used but wobble gives ~ 32 tRNAs with 20 specific synthetases. An amino acid's aminoacyl synthetase recognises specific identity elements in the appropriate tRNA molecule, often just 3 or 4 nucleotides. It has high specificity. tRNA with AA is said to be charged. Mature mRNA is ready to be transported to cytoplasm. mRNA also contains recognition sites for translation.
Ribosome: protein synthesis occurs when tRNA and mRNA associate with a ribosome, a biological machine that is more complex than the replisome. Bacterial cell contains about 10 000 ribosomes and eukaryotes many more. They comprise molecules of rRNA and ribosomal proteins in 2 subunits. The bacterial large subunit is 50S (sedimentation rate in glucose gradient): 23S rRNA (2900 nucleotides), 5S rRNA (120 nucleotides), about 34 proteins whereas the bacterial small subunit 30S: 16S rRNA (1540 nucleotides), ~21 proteins. Ribosome biogenesis - bacterial (e.g. E. coli): 16S:23S:5S genes in tandem together with tRNA genes. There are about 7 copies of the transcription unit that produces pre-RNA. Other RNases generate the correct ends of the molecules. Self assembly: 16S+21 ribosomal proteins => 30S subunit.    23S+5S + 31 ribosomal proteins => 50S subunit.
Ribosome biogenesis - eukaryotes   <   18S  >      <5.8S>        <    28S    >    ----> pre-18S+pre-(5.8S+28S) + 5S. Self assembly: 18S + 33 ribosomal proteins => 40S subunit. 28S+5.8S+5S+50 ribosomal proteins =>60S subunit. Ribosome structure - 50S subunit; has polypeptide exit and peripheral location of proteins (red). animation 
Initiation in prokaryotes: the process of synthesis if from NH2 to COOH. Energy is supplied by the high energy P bond with the tRNA. Small ribosome binds to sequence <6 bases all purines (AGGAGG - shine dalgarno sequence) in the 16S rRNA upstream of start codon AUG. Formylmethinone-tRNA binds to AUG together with initiation factors IF1, IF2 and IF3 to form initiation complex, IF3 released. 50S subunit binds to complex and forms two site for binding charged tRNA - P (peptidyl) and A (aminoacyl) - IF1 and IF2 released. Formylmethinone-tRNA is in P site guided by AUG on mRNA (sets reading frame).
Elongation second charged tRNA binds to A site matching next codon. Peptidyl transferase catalyses formation of peptide bond between AAs - part of 50S RNA. Bond between AA and tRNA in P site is broken. At this point, a dipeptide with tRNA attached in A site. Need to release the uncharged tRNA in the P site before continuing - passes through E site. mRNA-tRNA-AA1-AA2 complex moves towards the P site by width of 3 nucleotides due to conformational changes in the rRNA. Shift requires energy from GTP and several elongation factors (EFs). P site now occupied by a tRNA attached to peptide chain. Next charge tRNA moves into the A site. Shift requires energy from GTP and several elongation factors. Two EFs both increase speed and accuracy of synthesis - EF-Tu and EF-G bound to GTP.
Proposed role of EF-tu: charged tRNAs enter A site bound to EF-tu and EF-tu allows codon-anticodon binding but stops elongation until codon matching causes hydrolysis of bound GTP. The factor then leaves ribosomes without tRNA. It introduces delays before irreversible peptide formation. GTP hydrolysis is faster with correct codon-anticodon pairing and gives time for incorrect tRNAs-AA to exit. Dissociation of EF-Tu and full binding of tRNAs-AA to A site - incorrect base pairings are weaker and more liable to breakdown. Process is 99.99% accurate. The process is repeated as mRNA moves through the ribosome. After ~30 AAs, protein starts to emerge from hole in ribosome. Protein synthesis animation
Termination:  ribosome meets termination (stop codon) - UAG,UAA or UGA. Release factor (RF) not AA-tRNA in A site triggers action of GTP-dependent release factors that cut polypeptide from last tRNA e.g. RF-1 allows transfer to water, H2O instead of H2N-AA. Polypeptide splits from ribosome that separates into 2 subunits. Ribosome recycling occurs after termination (this is poorly urly understood).
Pollysomes: as the mRNA passes through the ribosome it can bind to new 30S subunits and initiate another polypeptide - polyribosomes.

Eukaryotic Translation:  large subunit 60S - 28S rRNA (4700 nucleotides) + ~49 proteins + 5S rRNA (120nucleotides) + 5.8S rRNA (nucleotides). Small subunit 40S - 18S rRNA (1900 nucleotides) + ~33 proteins mRNA lasts longer (hours) in eukaryotes. 5' end is capped and 3' end has poly A tail. It does not need formylmethionine. Also different sequence around AUG - ACCAUGG (Kozaki) - link. More protein factors are involved as well. Closed loop structure as protein factors link 5' cap and poly A tail. Protein is delivered directly into the endoplasmic reticulum. {Closed loop structure as protein factors link 5' cap and poly A tail. Positions 40S (small) subunit at 5' cap. Protein factors mRNA} (need picture here). Uses Met-tRNAi for initiation. Recognises the rRNA. Ribosome binding for initiation. 40S ribosome subunit binds to 5'cap + Met-tRNAi, then scans along AUG start codon where 60S binds. 
Some differences of protein factors between prokaryotes and eukaryotes: 
       EF-Tu + EF-Ts (prokaryotes) ---- EF1a + EF1βγ (eukaryotes)     role: binding aminoacyl-tRNA
              EF-G (prokaryotes)       ---- EF2 (eukaryotes)                    role: translocation
               RF-1 + RF2 (prokar.)    ---- eRF1 (eukaryotes)                   role: termination
              IF3 (prok.)                    ---- eIF3  (eukar.)                          role: prevents subunit reassociation
              

Antibiotics targeting different stages of protein synthesis in bacteria.
        Chloramphenicol  --- compepitive inhibitor of peptidyltransferase on 50S ribosome subunit
        Erythromycin      ---  inhibits peptidyltransferase on 50S subunit and inhibits translocation  - via 23S RNA
        Puromycin   ---    mimics 3' end of aminoacyl-tRNA
       Streptomycin ---  prevents correct codon: anticodon binding
       Tetracycline ---   binds reversibly to 30S subunit, also affects eukaryotic cells
    
Dipheria toxin acts on process of translation: the bacterial toxin enters the cell membrane. It requires Fragment B (membrane translocation). Fragment A (catalytic) is cleaved - enters cytoplasm leaving Fragment B in the membrane. Fragment A inactivates an Elongation Factor (EF2), the inactive EF2 inhibits protein synthesis. Once all the host's EF2 molecules are completed, the cell dies (OMG POOR CELLS). Protect by immunization. (for more info see also viral corruption of translation at section 10.16 of Craig: Molecular Biology: Principles of Genome function)

Summary and help:
  How we get from info in the genome store as a sequence of nucleotides in DNA to a functional protein molecule? Transfer of information through macromolecules. The central dogma of Molecular Biology: DNA is transcribed into RNA which is translated to Proteins. 
  Key events: transcription = RNA synthesis; translation = protein synthesis; genetic code = universal code for storage of info; mRNA processing = eukaryotes pre mRNA ->mRNA; post translational modification of proteins. All these lead from DNA to proteins to cellular function.
  Key players: 
         transcription: DNA template read in 3' to 5' direction. RNA polymerase. Synthesis of mRNA in 5' -> 3' direction. It requires promoters (and enhancers on DNA) and transcription factors. mRNA gets processed in eukaryotes.
         translation:  synthesis of proteins at ribosome, rRNA is an essential component. mRNA is the template carrying the genetic code and instructions for the ribosome of where to start. Code is 3 nucleotide codons specifying differnet amino acids -- colinear and non-overlapping, tRNA is an adaptor between nucleic acids and proteins - brings the correct amino acid into the ribosome by Watson-Crick base pairing its anticodon. 
     Mechanism: get the wider picture (central dogma - meanwhile be skeptical look at bruce lipton theories although not all may be correct, there may be some validity i.e. the central dogma is far more complex than current textbooks seem to depict involving DNA - RNA - Proteins to work back and forth regulating each other in a complex manner). Know the basics like key events and players. Watch and look for the animations and diagrams. They often summarize the processes and are much more memorable than the texts but extra detail is needed so access textbooks even journal articles. Put the mechanism in order - like a storyline (now comes the EF1 and later these ...). Build a picture of each process. Practice drawing diagrams and pictures of stuctures and processes - most helpful way to remember and learn.</sup>