Translation Notes

MRNA TRANSLATION

-Mature mRNA has a modified 5’ end which has a methylated guanosine residue attached by a 5’ to 5’ triphosphate linkage- this is attached to a cap binding protein- it also has a modified 3’ end which has a poly A tail and this is bound to a poly A binding protein

-these modifications protect from premature degradation, nuclear cytoplasmic export and the poly A tail facilitates in translation initiation

-There are also a 5’untranslated region and a 3’untranslated region- these regions contain regulatory elements that control how much mRNA is translated

-in between these untranslated regions- there is a coding region- which contains the sequence of nucleotides that specifies the sequence of amino acids in the polypeptide

Genetic code

-the mRNA is used as a template to translate the information into protein

-the sequence of 3 consecutive nucleotides on the mRNA strand, CODON codes for one amino acid- GENETIC CODE

-Each CODON codes for one amino acid

-there are four different nucleotides on the mRNA- therefore there are 64 possible combinations of 3 nucleotides- this means that the Genetic code is- REDUNDANT- some amino acids are coded by more than one triplet

-examples – AUG-codes for methionine and is the start codon from which translation begins

-UAA,UAG,UGA- are stop codons that are not recognised by tRNA molecules but proteins instead- these proteins force the termination of translation

-in theory, RNA sequence can be translated in any one of the 3 different reading frames- this depends on where the decoding process begins- different reading frames have a different sequence of codons-different sequence of amino acids-

-however only one of these three reading frames codes for the required protein- the genetic protein on the mRNA strand is read in non overlapping triplets from a fixed starting point- start codon AUG- Open reading frame consists of start codons that contains exclusively triplets that encode for amino acids

-deletion or insertation of a single or two nucleotides changes the sequence from that point onwards- frame shift mutations

Mutations and the genetic code

1. If a single nucleotide is changed

-Mis-sense mutation: the B globlin mRNA contains codon GAG-codes for Glutamic acid but when there is a substitution reaction a new codon is formed- GUG and this codes for valine- this leads to Sickle cell disease

-Nonsense mutation: E.g- B globin mRNA contains the codon- UGG however if the codon is changed to UGA it codes for a stop codon-causes Ribosomes to stop translation at the wrong place and a shortened protein with aberrant function or regulation is produced

Surveillance mechanism: nonsense mediated decay

• When splicing completes the exon-exon junctions are marked by the deposition of exon junction proteins

• These proteins serve as markers in indentifying faulty mRNA that contain premature stop codon- introduced due to a mutation in a gene or through mis-splicing

• The presence of a Exon junction protein down stream of a stop codon identifies the mRNA as faulty and induces its degradation

2. If single nucleotide is deleted

-if a number of nucleotides in an open reading frame are deleted/inserted and this number is not a multiple of 3- reading frame from that position onwards is changed- results in incorporation of incorrect amino acids into the polypeptide chain

-E.g- Huntingtons disease

Transfer RNA-tRNA

-small RNA molecules- 80 nucleotides long- base pairing occurs to form a clover leaf shape

-anticodon- three consecutive nucleotides that pairs with complementary codon in the mrna molecule

-single stranded 3’ region at the end of the molecule which is wher the amino acid that matches the codon is attached to the Trna at the carboxy end

-as there are 64 possible codons fromed from the 4 nucleotides- it would require 61 codons (3 stop codons)- however there are less than 45 t-RNAs- some of the t-RNAs can recognise more than one codon- these t-RNA differ in their third base- Trna molecules are constructed so they base pair with the first two bases of the codon and can tolerate mismatch with the third (wobble)- wobble base pairings mean less Trna molecules are needed.

Synthesis of Trna molecules

-Trna molecules are transcribed by RNA polymerase 3

-the enzyme- aminoacyl-Trna synthetases covalently couple a specific amino acyl AMPs-Activated amino acid (amino acid + ATP) to a single 3’ end of an appropriate set of Trna molecules- each enzyme is specific to the amino acid

-To ensure fidelity in the translation-each amino acid is matched to its codon through recognition nucleotides, anticodon in the tRNA

-this process is highly specific and proof reading ensures accuracy of the process

-this reaction is coupled with the hydrolysis of ATP to form a high energy ester bond between the amino acid and the 3’ end of the tRNA

Ribosomes: translation machinery

-protein synthesis occurs at the ribosome- consists of 50 different proteins, ribosomal RNA (Rrna)- RNA is the catalytic component: ribozyme

-the ribosome subunits are assembled in the nucleolus- the transcribed and modified Rrna associate with ribosomal proteins which are transported into the nucleus after their synthesis in the cytoplasm

-the two ribosomal proteins are then transported into the cytoplasm- these join together to initiate protein synthesis

-eucaryotic ribosomes- 80S- consists of one large- 60S, one small subunit-40S that fit together

-the small subunit provides the framework on which Trna can be attached accurately matched to the codons on the mRNA

-large subunit catalyses the formation of the peptide bonds that link amino acids together

-each ribosome has 4 binding sites- one for the mRNA and three (A-site (aminoacyl...

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