the genetic code Flashcards
what does understanding the genetic code allow us to do
-infer protein sequences from DNA sequence information.
-infer protein structure from its sequence.
-infer protein function by comparison with characterised proteins.
-design tools to study protein function, inhibitor drugs etc.
-The genetic code was determined before techniques were developed for the isolation of genes and sequencing of DNA.
what do tRNA ‘adaptor’ molecules read mRNA sequences through
-codon/anticodon interactions
-proteins made in the ribosomes are tRNA ‘adaptor molecules’
-Codons are read in a 5’ to 3’ direction and direct the protein synthesis in an N’ to C’ direction.
-mRNA codons are recognised by base-pairing with anticodon sequences within cognate tRNAs (should be attached to give AA)
-“Charged” tRNAs are aminoacylated: connected to the appropriate amino acid.
-Peptide bonds form between aminoacyl and peptidyl groups attached to adjacent tRNAs on the ribosome
PP chains extend by adding AA to C’ terminal end
how many reading frames does the coding sequence have
-3
-Genetic studies by Crick and Brenner showed that the genetic code was based on triplet codon sequences.
-All nucleotides have a genetic meaning; the code is nonpunctuated.
Multiple codons have the same meaning; the code is degenerate
-more codons than AA meaning more than one codon ca form the same AA-0 degenerate
the genotypes and its function (+ve or -ve)
wildtype= +
+1=-
-1=-
+1,-1=+
3(+1)=+
3(-1)=+
in vitro translation and ribosome binding assays
-Cell extracts can be “programmed” to make protein using artificial RNAs synthesized with polynucleotide phosphorylase- added radioactive labelled AA
-Poly(U) RNA was shown to specifically direct the incorporation of phenylalanine into protein. The codon UUU was deduced to encode phenylalanine.
-Binding of charged tRNAs to ribosomes in vitro is dependent upon the cognate trinucleotide codon.
-These experiments involved the enzymatic synthesis of nearly all 64 trinucleotides.
what’s the genetic code
-The genetic code is (almost) universal. All 64 codons have a meaning.
-61 sense codons encode a specific amino acid. There are 3 “stop” or termination codons.
-The “start” or initiation codon is (almost) always AUG. The N-terminal amino acid of proteins is methionine.
-Met and Trp are encoded by unique codons. Other amino acids are encoded by multiple codons; the code is degenerate.
-Synonymous codons (that encode the same amino acid) tend to differ at the 3rd nucleotide of the codon
-3rd nucleotide in codon is the least relevant
what occurs with the Non-Watson-Crick base-pairing at the 3rd base
-Some synonymous codons are recognised by different isoacceptor tRNAs (2 similar but different tRNA’s- recognise different codons) that are charged with the same amino acid- alternatively 1 tRNA can recognise more than one codon
-Other synonymous codons that differ at the 3rd base can be recognised by the same tRNA by wobble base-pairing.
-Many tRNAs have the nucleotide inosine (I) at the 1st position of the anticodon. I can base-pair with U, A and C. G can base-pair with C or U.
what structure does tRNA have
-cloverleaf secondary structure
-tRNAs have a “cloverleaf” structure. The 5’ and 3’ ends are drawn together. The amino acid is attached to the 3’ hydroxyl group of the 3’ terminal A nucleotide.
-All tRNAs have the 3’ terminal nucleotides CCA. These can be encoded within the genome (in E. coli) or added post-transcriptionally (in eukaryotes).
-Specific nucleotides within tRNAs are post-transcriptionally modified. About 10% of nucleotides in tRNA are modified.
-Modification of the 1st position of the anticodon allows “wobble”.
what shape are tRNA’s folded into
-L-shape
-Coaxial stacking of short helices within RNA increases its thermodynamic stability.
-The acceptor stem is stacked on the TYC arm. The anticodon is stacked on the D arm. Base-pairing occurs between the D and TYC loops.
-The interactions produce a flat, L-shaped molecule where the anticodon loop and aminoacyl group are positioned at opposite ends of the molecule.
what are Charging tRNAs
-tRNA charging is mediated by aminoacyl-tRNA synthetases- The reaction requires ATP.
-There are 20 different aminoacyl tRNA synthetases. A single enzyme charges all isoacceptor tRNAs.
-The amino acid is linked to tRNA by an ester linkage between the carboxylic acid group of the amino acid and the 3’ hydroxyl group of the terminal nucleotide.
