Translation Flashcards
Describe the genetic code
The linear sequence of nucleotides in DNA is transcribed into mRNA which specifies the linear sequence of amino acid residues in the protein.
The sequence of nucleotides in DNA that specifies the amino acid sequence in a protein is called the GENETIC CODE.
The genetic code has 64 triplet codons that specifies for the 20 standard AA
Explain the features of the genetic code
- It consists of non overlapping triplet codons
- It is universal.
- It is unambiguous. Each codon corresponds to only one amino acid.
- It is generative. There are several codons for most amino acids. For example, there are 6 for serine, 4 for glycine and 2 for lysine. This minimizes the effects of mutations since a change in a single nucleotide often produces a codon that specifies for the same amino acids.
- There several codons that specify for some amino acids and such codons are called synomynous codons. The 1st 2 letters are the same and the 3rd letter is variable. For example the amino acid glycine is specified by GGU,GGC,GGA and GGG
Describe the structure of tRNA
The tRNA has a cloverleaf structure and it has between 75 to 95 nucleotides.
It has an anticodon loop that has the 3 bases that determine the type of amino acid that will attach to the tRNA.
The mRNA and tRNA interact through the base pairing between the codons and the anticodon.
The anticodons of many tRNA can pair with more than one codon. The anti codon can pair with the 1st and 2nd base of the codon while the 3rd base is flexible. The 3rd position is called the wobble position
What is translation?
The mRNA produced in the nucleus in the process of transcription is transported in the cytosol to the ribosomes which are the site of protein synthesis.
The synthesis of proteins from the information that is encoded in mRNA is called translation.
List the stages of protein synthesis
i) Activation of the AA.
ii) Initiation,
iii) elongation,
iv) termination,
v) post translation modification.
Describe stage 1 of protein synthesis
Stage 1: Activation of Amino Acids
* Two fundamental chemical requirements must be met:
(1) the carboxyl group of each amino acid must be activated to facilitate formation of a peptide bond, and
(2) a link must be established between each new amino acid and the information in the mRNA that encodes it.
* Both these requirements are met by attaching the amino acid to a tRNA.
(Takes place in the cytosol, not on the ribosome)
* Each of the 20 amino acids is covalently attached to a specific tRNA at the
expense of ATP, using Mg2
⁺- dependent activating enzymes known as
aminoacyl tRNA synthetases.
* When attached to their amino acid (aminoacylated) the tRNAs are said to be
“charged.
Describe stage 2 of translation
Initiation is divided into 3 steps:
* Step1. the ribosome(70S) separates into the 2 subunits, 30S and 50S. The 30S subunit has got 2 sites the P site and the A site. The 30S ribosomal subunit binds two initiation factors, IF-1 and IF-3. The IF-1 prevents the premature binding of tRNA to mRNA while IF-3 prevents the 30S and 50S subunits from combining prematurely.
* The mRNA then binds to the 30S subunit.
* The initiating (5’)AUG is guided to its correct position by the Shine- Dalgarno
sequence in the mRNA.
* In step 2, the complex consisting of the 30S ribosomal subunit, IF-3, and mRNA is
joined by both GTP-bound IF-2 and the initiating fMet-tRNAfMet. The anticodon of this
tRNA now pairs correctly with the mRNA’s initiation codon.
* In step 3 this large complex combines with the 50S ribosomal subunit.
* Simultaneously, the GTP bound to IF-2 is hydrolyzed to GDP and Pi, which are released
from the complex.
* All three initiation factors depart from the ribosome at this point.
List the protein factors required for initiation of translation in bacteria and state their respective functions
IF-1 : Prevents premature binding of tRNAs to A site
IF-2 : Facilitates binding of Met-tRNA** to 30S ribosomal subunit
IF-3 : Binds to 30S subunit; prevents premature association of 50S subunit; enhances specificity of site for fMet-RNA et
List the protein factors required for initiation of translation in eukaryotes and state their respective functions
- eIF2 : Facilitates binding of initiating Met-tRA™* to 405 bosomal subunit
- eIF2B, eIF3 : First factors to bind 40S subunit; facilitate subsequent steps
- eIF4A : RNA helicase activity removes secondary structure in the mRNA to permit binding to 40S subunit; part of the elf complex
- eIFB : Binds to mRNA; facilitates scanning of mRNA to locate the first AUG
- eIF4E : Binds to the 5’ cap of mRNA; part of the elFF complex
- eIF4G : Binds to elF4 and to poly(A) binding protein (PAB); part of the elFF complex
- eIF5 : Promotes dissociation of several other initiation factors from 40S subunit as a prelude to association of 60S subunit to form 80S initiation complex
- eIF6 : Facilitates dissociation of inactive 80S ribosome into 40S and 60S subunits
Describe stage 3 of protein synthesis
Elongation occurs in 3 steps
Step 1: Binding of an Incoming Aminoacyl-tRNA
In the first step, the appropriate incoming aminoacyl-tRNA binds to GTP which is bound to EF-Tu.
The resulting aminoacyltRNA– EF-Tu–GTP complex binds to the A site of the 70S initiation complex.
The GTP is hydrolyzed and an EF-Tu–GDP complex is released from the 70S ribosome.
The EF-Tu–GTP complex is regenerated in a process involving EF-Ts and GTP.
Step 2: Peptide Bond Formation
* A peptide bond is now formed between the two amino acids bound by their tRNAs to the A and P sites on the ribosome.
* The α-amino group of the amino acid in the A site acts as a nucleophile, displacing the tRNA in the P site to form the peptide bond.
* This reaction produces a dipeptidyltRNA in the A site, and the now “uncharged” tRNAfMet remains bound to the P site.
* Peptidyl transferase catalyzes peptide bond formation. This reaction is catalyzed by the 23S rRNA.
Step3: Translocation
Movement of the ribosome along the mRNA requires EF-G (also known as translocase) and the energy is provided by hydrolysis of another molecule of GTP.
The ribosome, with its attached dipeptidyltRNA and mRNA, is now ready for the next elongation cycle and attachment of a third amino acid residue.
This process will continue until the whole mRNA is translated
How is the elongation stage in eukaryotes similar to that in prokaryotes?
The elongation cycle in eukaryotes is quite similar to that in prokaryotes.
Three eukaryotic elongation factors (eEF1α, eEF1βγ, and eEF2) have functions analogous to those of the bacterial elongation factors
(EF-Tu, EF-Ts, and EF-G, respectively).
Eukaryotic ribosomes do not have an E site; uncharged tRNAs are expelled directly from the P site.
Describe stage 4 of translation
Stage 4: Termination of Polypeptide Synthesis
Termination is signaled by the presence of one of three
termination codons in the mRNA (UAA, UAG, UGA).
Releasing factor RF1 recognizes that a stop codon
resides in the A site.
RF1 is bound by a complex consisting of releasing factor RF3 with bound GTP.
This complex, with the peptidyl transferase, promotes
hydrolysis of the bond between the peptide and the
tRNA occupying the P site
Describe stage 5 of translation
Stage 5: Folding and Posttranslational Processing
* In order to achieve its biologically active form, the new polypeptide must
fold into its proper three-dimensional conformation.
* Before or after folding, the new polypeptide may undergo enzymatic
processing,
removal of one or more amino acids;
addition of acetyl, phosphoryl, methyl, carboxyl, or other groups to certain
amino acid residues;
proteolytic cleavage;
attachment of oligosaccharides or prosthetic groups
Which drugs/toxins inhibit protein synthesis?
- Puromycin - Its structure is very similar an aminoacyl-tRNA, enabling it to bind to
the ribosomal A site and participate in peptide bond formation, producing
peptidyl-puromycin. It does not engage in translocation and dissociates from the ribosome. This prematurely terminates polypeptide synthesis. - Tetracyclines inhibit protein synthesis in bacteria by blocking the A site on the
ribosome, preventing the binding of aminoacyl-tRNAs. - Chloramphenicol inhibits protein synthesis by bacterial ribosomes by blocking
peptidyl transfer; it does not affect cytosolic protein synthesis in eukaryotes. - Cycloheximide blocks the peptidyl transferase of 80S eukaryotic ribosomes but
not that of 70S bacterial (and mitochondrial and chloroplast) ribosomes. - Ricin, an extremely toxic protein of the castor bean, inactivates the 60S subunit
of eukaryotic ribosomes by depurinating a specific adenosine in 23S rRNA. - Streptomycin, a basic trisaccharide, causes misreading of the genetic code (in
bacteria) at relatively low concentrations and inhibits initiation at higher
concentrations. - Diphtheria toxin catalyzes the ADP-ribosylation of a diphthamide (a modified
histidine) residue of eukaryotic elongation factor eEF2, thereby inactivating it.
