The Genetic Code and tRNA Flashcards
Why is protein synthesis so costly (takes a ton of ATP)
-Biochemically challenging
-Takes more biosynthetic pathways
-Folding is challenging
How was it decided that there is a triplet code?
-There are 4 nucleotides and 20 amino acids
-Amino acid pairs = 4^2 = 16 which is less than 20 amino acids, meaning that there wouldn’t be enough codes to code for them all
-4^3 = 64 which is more than 20
What were the resulting questions from realizing the triplet code?
-There are 20 amino acids but 64 codons
-Meaning either 44 codons don’t code for an AA of multiple codons specify the same amino acid
-How is the sequence grouped? Overlapping or non-overlapping?
What does it mean to have an overlapping or non-overlapping codon
-Non-overlapping is sequential like we have today, after the first three are read, you move on to the next three
-Overlapping would mean that you read the first three as a codon, then move everything over by one so you’re reading starting with the second nucleotide in the sequence
George Gamow speculation
-Holes in the major groove of DNA
-Maybe amino acids pop in there and link together over time
-Would require cross talk to the other side of the major groove
-Free amino acids get caught in the holes, which unites the peptide chains
What are important characteristics of the genetic code?
The genetic code is nearly:
-Universal: conserved in nearly all forms of life
-Non-overlapping: translated sequences do not overlap with their neighbors
-Degenerate: there is some redundancy, such as there being 64 codons for 20 amino acids
-Triplet: three nucleotides of RNA encode 1 amino acid
The triplet of RNA that encodes one amino acid is called a codon, what does this mean for nucleotide mutations?
-There is a reading frame that triplets appear in
-Different kinds of nucleotide polymorphisms (deletions, substitutions, insertions) have different impacts. Some are completely silent
How can mutations affect the reading frame
-Addition or deletion of 1 or 2 bases causes a frameshift, resulting in junk
-Addition or deletion of multiple of 3 can still specify a functional protein
How was the genetic code deciphered (experimental set up)
-Cell-free in vitro translation
-E. coli extract treated with DNase (to degrade any DNA)
-mRNA eventually degrades too (so no mRNA left over from original cell)
-Synthetic homo-polymers of NTPs (RNA), form chain of RNA with same nucleotide
-Incubated this with AAs, with one labeled radioactively while the others are unlabeled
—–Had 21 tubes, one tube for each radioactively labeled amino acid, plus control with no radioactive labeling
How was the genetic code first deciphered (actual experiment)
-First try was with poly(U)
-Produce synthetic homo-polymers of NTPs (RNA) (chain of U NTPs)
-Incubate poly(U) with mixture of 1 radioactive and 19 unlabeled AAs, plus ATP and GTP
-Radioactive protein only appeared when phenylalanine (Phe) was labeled
-Allowed for UUU to be determined to be codon for Phe
-Repeated with poly (A) and poly(C)
-Did not work with poly(G) because it formed a structure that didn’t allow it to form a chain (guanosine tetraplex)
How were the rest of the codons assigned to amino acids? How were reading frames seen?
-Khorana
-Building smaller building blocks, such as dinucleotides and trinucleotides, and linking them together
-Result was different amino acids
Ex: UAC repeated resulted in tyrosine, theronine, or leucine (but only the one depending on the reading frame, like Tyr-Tyr-Tyr or Thr-Thr-Thr)
-Turns out that depending on reading frame, they would get different amino acids
How were stop codons discovered?
-Tetranucleotides
-Now you get dipeptides and tripeptides, but then you would get a stop codon
-So basically you would get chains of two or three peptides, and then it would just stop
-Determined that there were stop codons
What amino acids only have one codon?
Met and Trp
What does it mean for codons to be synonymous?
-Amino acids are specified by multiple codons
-Degenerate because of redundancy
How is the arrangement of the table non-random
-Because you have sequences of nucleotides encoding different amino acids, codons have a propensity to mutate to some amino acids over others
-Mutations are more likely to mutate to residues that are similar to them than drastically change, such as hydrophobic residues being more likely to mutate to other hydrophobic residues
-Buffering
