BioMG 3320 1st Edition Lecture 3Outline of Past LectureI. Alterations in DNAII. Definition of GeneIII. Bacterial CellsIV. Eukaryotic CellsV. Transposons VI. Human GenomeOutline of Current LectureI. Solving the Genetic CodeII. Experiments to Determine Genetic CodeIII. More on the Genetic CodeIV. Degeneracy in the CodeCurrent LectureI. Solving the Genetic Code- There are two types of info contained in DNA-Coding information-Regulatory information- Basics of translation into a protein-Ribosome attaches to the mRNA on the 5’ end and moves to the 3’ end-As the ribosome moves, it decodes the sequence of base pairs into amino acids These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.-The amino acid is then added to the chain, forming a sequence that makes a protein- Four base pairs encode for twenty amino acids-There has to be more than 16 possibilities (42)-There must be at least three base pairs per amino acid-The code is collinear: the nucleotide order corresponds to the amino acid order- Is the code non-overlapping or overlapping?-Experiments were conducted to determine thisII. Experiments to Determine Genetic Code- At first, researchers had a huge collection of mutants that destroyed protein. They foundthat an insertion mutation as well as a deletion mutation caused an effect on the entire sequence, suggesting that the code must be non-overlapping-If it was overlapping, the effect would only be localized-It is possible to have both an insertion and deletion mutation together, and have the reading frame restored- In one experiment, Nirenberg and Khorana took cell extract, ATP and one amino acid-They had poly(U) + phenylalanine and they got a polypeptide-When they tried poly(U) + other amino acids, they did not get a polypeptide-Suggests that UUU codon encodes phenylalanine-They did this for numerous other amino acids paired with codons as well-However, random polymers of more than one nucleotide encode multiple codons so needed further experiments - In the second experiment, they made a random polymer using five times more A than C-They found that AAA was the most common codon and CCC was the rarest-They determined how many times specific amino acids were observed-They were then able to match the amino acid with the code based on the frequency of the amino acid and how common the code was present - In the third experiment, Khorana created synthetic RNA with defined sequences (not random)-Example: (AC)n -They found that two amino acids formed: Threonine and Histidine-So ACA encodes for Thr and CAC codes for His OR ACA codes for His and CAC codes for Thr-Based on other experiments, His known to be AC2 so CAC codes for His and ACA codes for Thr- A confirmation of these results:-Purified ribosomes and synthetic RNA bind SPECIFIC aminoacylated tRNA III. More on the Genetic Code- There is a redundancy in the code. Many amino acids have more than one codon- 61 codons encode amino acids- Stop codons: UAA, UGA and UAG (only function is to stop translation)- Start codon: AUG-This codon has two functions: start translation if in beginning of sequence and add Met if in middle of sequenceIV. Degeneracy in the Code- Some mutations will not change the encoded amino acid sequence-Synonymous: no change in encoding-Non-synonymous: change in encoding-Example of synonymous: ACA, ACU, ACC, ACG all encode Thr; a change in sequence at position three of any Thr codon does not alter encoding- Insertions and deletions (that are not multiples of three nucleotides) will completely alter the encoded amino acids 3’ of the mutation: frameshift mutation- Mutations that do not change the amino acid sequence can have an effect- Degenerate codons are not used equally. Codons used frequently correspond to tRNAs present at higher levels; codon bias differs between species-If rare codons are used, it is difficult to efficiently translate the mRNA- tRNA has an anticodon which recognizes codon -Aminoacyl-tRNA synthetases recognize the correct tRNA and covalently attach the correct amino acid- Wobble pairing-There are more codons than tRNAs which means some bases can pair with multiple other bases (EX: G can pair with C or U) so tRNA can recognize multiple combinations-Inosine is a modified base that has the ability to pair with various bases- Variations in the genetic code in mitochondria:-Genetic code is almost universal so a change in the code is catastrophic because it will affect all proteins-However, mitochondria encode very few proteins so that is why there can be variations in the genetic code- 21st amino acid-Certain rare proteins require selenocysteine for function-Selenocysteine tRNA is specific for selenocysteine. It recognizes UGA and recruits special machinery to recode this codon so that Ser is converted to selenocysteine- RNA editing-Covalent alteration of messenger RNA-Cell makes transcript, special enzymes recognize and covalently change some of the base pairs (deamination)- One gene can have two products if the stop codon is in a different place-apoB gene in liver makes protein 100 kD but apoB gene in intestine makes protein 48 kD-This gene has different functions in the two different locations, so the expression is different-The stop codon is placed earlier in the intestine than it is in the