Lecture 8 Where are we in the Central Dogma? Flashcards
why is translation important?
Translation is, perhaps, the single most important event in biology because what protein is translated versus what isn’t translated makes the difference between your body building a heart versus lungs.
why is protein synthesis important?
..no field has occupied more investigators in the biological sciences than protein synthesis. The subject permeates almost all parts of biology, from translation itself to regulation of gene expression ….and the origin of life.
Steps toward an in vitro Protein Synthesis System
Cancer research and (peaceful) approaches using radioactivity drove research into protein synthesis
Mostly performed on higher animals rather than bacteria
1953-first cell free system capable of carrying out peptide bond formation using 14C-amino acids.
Demonstrated the activation of amino acids by formation of aminoacyl adenylates from amino acids and ATP.
Zamecnik and Hoagland noticed that the RNA became labeled with 14C-amino acids and that the labeled RNA was subsequently able to transfer the amino acids to protein.
The transfer was dependent upon guanosine triphosphate.
Protein Synthesis via translation of the mRNA code
Protein synthesis occurs via translation because the “language” of the nucleotide sequence on the mRNA is translated into the “language” of an amino acid sequence.
All ingredients for protein synthesis are coded by the DNA, some remain as RNA (such as tRNA, rRNA), others become translated into proteins (such as mRNA).
Ribosomes = rRNA + ribosomal proteins
Recipe for mRNA translation/protein synthesis:
mRNA directs translation by carrying the genomic code from DNA
tRNA reads the genetic code and brings the right amino acid to the site of translation.
Ribosomes align tRNA on mRNA and fuse amino acids together to make the polypeptide.
Genetic code is a triplet code
The genetic code is a language.
The four bases (A,U,G,C) are the letters of this language.
tRNA is the reader of this language.
The Triplet Codons form the genetic code
There are, therefore, 4 x4 x 4 = 64 different combinations of bases and therefore 64 triplet codons (or words) in mRNA genetic language.
61 of the 64 codons code for the 20 common amino acids.
Characteristics of the genetic code
Specificity. A particular codon always codes for the same amino acid. e.g. CGU is the codon for arginine.
Degeneracy. A given amino acid may be specified by more than one triplet codon. e.g. Leucine is specified by six different codons.
Only Met and Trp have just one coding triplet.
A nearly Universal code. A codon specifies the same amino acid in all organisms.
Universality of genetic code allows codons with DNA vaccines/mRNA vaccines to make the same protein as would be made in the natural hosts of the pathogens (viruses) or within the pathogens themselves (bacteria).
Nonoverlapping and comma-less: Once the reading is commenced, the code is read from a fixed starting point as a continuous sequence of THREE bases at a time (CODON) until STOP codon is reached. For example, AUACGAGUC is read as AUA/CGA/GUC without any “punctuation” between the codons.
Removal or addition of a base can have detrimental effect on protein structure
Actual reading frame dictated by AUG start codon.
Frameshift mutations caused by deletions that disrupt reading frame.
Rarely generate functional protein.
Mutations can have pathological consequences in humans
In sickle cell anemia one single substitution of a T to A converts CTC (codon for glutamic acid) into CAG (codon for ???) in the β-globin gene changes the shape of the red blood cells.
Mutations can confer antibiotic resistance in bacteria
Amino acid changes and missense mutations in the rpoB gene confer resistance of Mycobacterium tuberculosis to rifampicin (targets RNA polymerase).
These changes can also have fitness costs to the bacteria.
Summary part one
Translation is important!
Genetic code is contained within the mRNA and is read by tRNA with ribosomes.
Genetic code is (almost) universal and changes in the code may impact on the protein sequence and have implications in the phenotype.
Changes in the code (point mutations, single nucleotide polymorphisms) can result in diseases and also confer antibiotic resistance.
Steps in Protein Synthesis (Translation)
1.Activation. Coupling correct amino acid to appropriate tRNA molecule (tRNA charging).
2.Initiation: Assembly of the ribosome subunits, the initiator tRNA and mRNA.
3.Elongation: Amino acids added to nascent polypeptide chain in cycle.
4.Termination: Release of polypeptide chain from tRNA and dissociation of ribosomes.
tRNA
~70 to 80 nucleotides long
Cloverleaf structures
The adaptor function-mRNA template is recognized by the anticodon loop and binds to the appropriate codon by complementary base pairing (wobbly)
Anticodon loop of tRNA has anticodon base triplet, which is complementary to the codon in mRNA.
Amino acid attached to conserved sequence CCA at 3′ terminus.
Activation: Aminoacyl-tRNA Synthetases charge tRNA with right amino acid
20 different enzymes (why 20?).
These enzymes generate a high energy ester bond.
This ensures that tRNA is charged with correct amino acid.
Ribosomal subunits are vital for translation
Ribosomes binds to the mRNA via the small subunit.
Once the connection with mRNA-small subunit is established, the large subunit can bind.
The humble ribosome
Ribosomes-classified according to rates of sedimentation
70S=bacterial ribosomes
80S=eukaryotic ribosomes.
Two distinct subunits composed of proteins and rRNAs.
Cells typically contain many ribosomes
-Escherichia coli ~20,000 ribosomes (25% of the dry weight of the cell)
-mammalian cells ~10 million ribosomes.
Each ribosome: one copy of the rRNAs and one copy of each of the ribosomal proteins, with one exception which is present in four copies.
The subunits of eukaryotic ribosomes are larger and contain more proteins than their prokaryotic counterparts have.
The small subunit (40S) of eukaryotic ribosomes is composed of the 18S rRNA and approximately 30 proteins; the large subunit (60S) contains the 28S, 5.8S, and 5S rRNAs and about 45 proteins.
“Geography” of mRNA
5’UTR- (From
+1 to start codon)
3’ UTR-(From
Stop codon to Tc terminator)
How does ribosome bind to the mRNA?
Prokaryotes: the rRNA component the 30S ribosomal subunit binds to the Shine-Dalgarno sequence within the mRNA
In eukaryotes, 5’ Cap helps align the ribosome on mRNA
RNA Binding Sites in Ribosome
A = aminoacyl-tRNA enters
P = peptidyl-tRNA (location of polypeptide)
E = tRNA exits leaving amino acid for translation
INITIATION
Binding of tRNA and mRNA with Ribosome.
Requires IF1, IF2, IF3 factors.
IF1 and IF3 attack 70S ribosome and dissociate 30S subunits from 50S preventing premature assembly.
30S lands onto the mRNA, Shine-Dalgarno sequence help align 30S.
IF2 brings initiator tRNA at P (peptidyl) site, tRNA binds to AUG codon through base pairing.
IF2 is released, IF1 and IF3 also released to allow 50S subunit to form a mRNA:tRNA:ribosome complex.
ELONGATION
At the beginning of each elongation cycle the polypeptide chain is attached to peptidyl tRNA.
EF-Tu escorts new charged tRNA to A (aminoacyl) site.
Peptidyl transferase activity within 50S subunit catalyses peptide bond formation.
A peptide bond is formed between the last amino acid in polypeptide.
EF-G pushes ribosome ahead on mRNA allowing deacylated tRNA to leave through E (exit) site, causing A site to be ready to accept next charged tRNA.
TERMINATION
The completion of polypeptide synthesis is signalled by the translocation of one of the stop codons (shown in red).
Because there are no tRNAs to recognise stop codon, protein release factors participate in the termination process to stimulate the release process.
Several antibiotics work by targeting translation
Kanamycin
-Produced by Streptomyces griseus.
-Binds to 30S and distorts its structure, interfering with initiation.
-First aminoglycoside antibiotic described in 1944
-Streptomycin was used as a monotherapy regimen to treat TB.
-Other aminoglycosides very important antibiotics e.g. gentamicin, amikacin, tobramycin, neomycin, plazomicin, paromomycin
