Lecture 6 Flashcards
How many RNA bases per codon?
reading mRNA 3 bases at a time is logical, because:
Codons with 1 or 2 bases can’t make enough different “words”
Codons with 4 bases seems inefficient/wasteful (=256 “words”!)
Brenner and Crick
worked with mutagens that cause single
base-pair insertions or deletions in DNA.
Overlapping or non-overlapping?
didn’t know whether the reading frame shifts to three
brand-new nucleotides for each codon (non- overlapping),
or if it only shifts 1 base (for e.g.), so that each codon overlaps with the codons read before and after it (overlapping).
Because these particular kinds of point mutations only affect one amino acid, we know that our genetic code is nonoverlapping.
Which amino acid each 3-base codon codes for?
- We can mix RNA nucleotides together in vitro with an enzyme (polynucleotide phosphorylase) to randomly make RNA strands.
- We can translate RNA strands in vitro too (so no cells or AUG needed… the whole strand gets translated into a polypeptide).
Procedure for “cracking the code”
- Mix Uracil (U) with the enzyme, to make a poly(U) strand.
- Take the results of 1. and add ribosomes and all amino acids
- See what amino acids are joined together, to determine that UUU codes for Phenylalanine (Phe)
- Repeat with other nucleotides to figure out that AAA = lysine, GGG = glycine, and CCC = proline. That’s 4 done!
Second Part of procedure
- Mix enzyme with 2 kinds of nucleotides (at different amounts)
- Take the results of 1. and add ribosomes and all amino acids
- So we’ve identified all 6 amino acids that can be encoded with just A and C… but we can actually narrow it down even further using probability calculations…
Gorbind Khorana
-Gorbind Khorana took AC dinucleotides and joined them to make ACACACACACACACACAC, or (AC)n.
-This gives us alternating ACA and CAC codons.
-Translation gave us alternating Thr and His.
-So NOW we know that ACA= Thr and CAC = His
So logically combining these 3 experiments in different combinations gives us our full genetic code!!
the genetic code
-We begin translation at the start codon (AUG), and we read every 3-base codon consecutively (with no overlap and no skipping of bases) until we get to one of the stop codons.
in vitro: whole mRNA was translated by ribosomes in a test tube
in vivo: starting translation at AUG sets the right reading frame.
add title 2
- The genetic code is almost universal (i.e. shared by all life).
- This is why we can add a human gene into a bacterial
plasmid. and the bacteria can make the correct protein for us. - The code is degenerate: most amino acids have more than one codon that codes for it (like “synonyms”)
- The code is unambiguous: each codon only encodes one type of amino acid. (each word refers to just one “ingredient”)
- There are 3 stop codons: UAA, UAG, UGA.
- The other 61 codons are sense codons that code for specific amino acids.
redundancy
this degeneracy doesn’t affect the cost of making the DNA or RNA (e.g. making UUU requires similar costs as UUC).
the expected cost of making enough tRNAs to match all 61 sense codons is less than you think! (don’t need 61 tRNAs!)
e.g. If a tRNA has an AAG anticodon, it would bind with the UUC codon and bring Phe.
Imagine that the third letter of the AAG is less ‘picky”, and could bind to U as well. Then we would only need one tRNA for both Phe codons!
Wobble Hypothesis
First 2 codon positions must be perfectly complementary (very “picky”); but sometimes position 3 of the codon will pair with non-complementary bases (less “picky”)
(i.e. the binding at this 3rd “wobble” position is looser)
Therefore, we don’t need 61 tRNAs to translate all 61 codons!
mutations
So if you know that a point mutation occurred near the beginning of the coding sequence, then you can
expect an amino acid near the N-terminus of
the polypeptide to be affected.
Point mutation
a general term for a change in a single complementary base pair in the DNA.
e.g. Base insertions and deletions that cause a frameshift. base substitutions don’t cause frameshifts,
but they can affect the polypeptide in different ways
Nonsense mutations
often create short, non-functioning proteins, since a new stop codon (e.g. UAG) ends translation early.
However, a suppressor mutation can allow the whole gene to be translated again. How can a point mutation suppress the effects of nonsense mutation?
e.g. Mutate a tRNA gene so the anticodon becomes AUC!
Post-translational processing of polypeptides
Gene expression isn’t finished until the protein is fully formed and functional. To get there, the polypeptide may undergo:
Cleavage: e.g. a piece of a precursor polypeptide might be cut off to make the active form of the protein.
Folding: e.g. a polypeptide may need “chaperone” proteins to help them fold properly. (Misfolded proteins get degraded)
Bonding to other polypeptides: e.g. to make polymers.
Modification of side chains: e.g. phosphorylation by kinases, or glycosylation (attachment of sugar molecules).
Localization: e.g. being moved to a particular organelle.
