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Chapter 32
The Genetic Code
Including a review of tRNA structure
from Chapter 12 section 7
Review of Steps in Gene Expression
RNA = nucleotide sequence
Adaptor molecule
protein = amino acid sequence
From Access Excellence: http://www.accessexcellence.org/AB/GG/steps_to_Prot.html
Translating the Message
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How does the sequence of mRNA translate into the
sequence of a protein?
What is the genetic code?
How do you translate the "four-letter code" of mRNA
into the "20-letter code" of proteins?
And what are the mechanics like? There is no
obvious chemical affinity between the purine and
pyrimidine bases and the amino acids that make
protein.
Three major advances gave the clues to solving this
dilemma
Clue #1
The Discovery that Proteins are made on
ribosomes
• By Paul Zamecnik (early 1950s)
• He asked where in the cell are proteins
synthesized?
• Injected rats with radioactive amino acids
• A short time after injection (when the amino acids
should be incorporated into newly-synthesized
proteins) he killed the rats, harvested their livers,
ground them up and divided the cell components
into “subcellular fractions” by centrifugation
Results
• Radioactivity was found in small ribonucleoprotein
particles visible by electron microscopy.
• These were later characterized and called
“ribosomes” (since they had RNA as a major
component)
From Lehninger “Principles of Biochemistry” p 1021
Clue #2
The Discovery that amino acids are
“Activated”
• By Hoagland and Zamecnik
• They incubated amino acids with the cytosolic fraction
of liver cells, and with ATP
• They found the amino acids became “activated” during
the incubation
• Activation consists of attaching the amino acids to a
heat-stable soluble RNA (which we now know is tRNA)
• Activated amino acids are called aminoacyl- tRNAs
• The enzymes that do the activation are called
aminoacyl-tRNA synthetases (next class!)
Clue #3
Crick’s Adaptor Hypothesis
• Francis Crick thought about the problem
• He reasoned that a small nucleic acid could serve as
an adaptor between RNA and protein synthesis if it
could bind both RNA and an amino acid
• His idea was that one end of the adaptor would bind a
specific amino acid and the other would bind to a
specific sequence in the RNA that coded for that amino
acid
Crick’s Adaptor Hypothesis
•Must have one adaptor
per amino acid
•Therefore there must be
a family of adaptors:
•These are the tRNAs
• each tRNA can
recognize specific
sequences in the RNA
transcript
•Each is “charged” with
the amino acid that is
specified by that sequence
From Lehninger “Principles of Biochemistry” p 1021
Review of tRNA Structure
• tRNAs are the “adaptors” in protein synthesis
• There are many different tRNAs, each has a distinct
sequence
• However, all tRNA have several conserved features
1) small (73-93 nucleotides long)
2) they have a conserved secondary structure - 4
stems and 4 loops with important functions
3) they contain many unusual bases
Inosine (I), pseudouridine (y), dihydrouridine (D),
ribothymidine (T), and methylated bases (mG, mI)
Unusual bases in tRNAs
Invariant bases
Amino acid
addition site
Interacts with
the ribosome
Varies in size
Base pairs with the codon
in the mRNA transcript
Example of a
specific tRNA
The yeast
alanyl-tRNA
The cloverleaf tRNA
folds into an L-shape by
interactions between
conserved bases in the
stems and loops
tRNA tertiary structure: banana or L-shape
Noncanonical base pairs stabilize the 3o structure
Animation of tRNA secondary and
tertiary structure
Elucidation of the Genetic Code
4 major advances helped figure out the code
1) The demonstration of colinearity
between genes and protein
2) The idea of triplet codons
3) Deciphering the first word (UUU= Phe)
4) Deciphering the rest of the code
The Colinearity of Gene and
Protein Structures
• Yanofsky provided evidence in 1964: he showed
that the relative distances between mutations in
DNA were proportional to the distances between
amino acid substitutions in E. coli tryptophan
synthase
Charles Yanofsky - 1964
(trp operon)
• Had a collection of mutants in the trpA gene (coding for
tryptophan synthase)
• He made 2 determinations using these mutants:
1) determined the position of the mutation in the gene
2) determined the position of the mutant amino acid in the
protein encoded by each mutant gene
Mutant 1
Mutant 3
Mutant 2
gene
protein
Therefore gene sequence is colinear with protein sequence.
What is the nature of the Code
mRNA (nucleotides)
4 different nucleotides
protein (amino acids)
20 different amino acids
• 1:1 correspondence can’t work
• Therefore nucleotides must be read in combinations
• Is 2 enough? 4X4 = 16 different combinations possible
- not enough
• But 3 would give 4X4X4 = 64 combinations
• This would be enough to code for 20 amino acids
• Therefore the concept of the triplet codon was born
What is the nature of the Code
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Is the code overlapping or non-overlapping?
Is the code punctuated or non-punctuated?
These details were worked out by Crick
Crick used mutagens to introduce changes
into a coding sequence
• Used mutagens that either induced point
mutations in the DNA or insertions.
• After generating mutants he checked the
proteins coded by the mutant sequences
Changes 3 amino
acids
X
X
Changes 1 amino acid
Changes all following
amino acids
following amino acids
are unchanged
The Nature of the Genetic Code
summary of results
• A group of three bases codes for one
amino acid
• The code is not overlapping
• The base sequence is read from a fixed
starting point, with no punctuation
• The code is degenerate (in most cases,
each amino acid can be designated by
any of several triplets)
Biochemists Break the Code
Assignment of "codons" to their respective amino acids
• Marshall Nirenberg and Heinrich Matthaei
Worked with an in vitro translation system from E. coli
Cell-free extract
•Ribosomes
•tRNAs
•Amino acids
•Enzymes
•ATP, GTP
+ mRNA = protein
Deciphering the first word
They generated RNAs that are homopolymers
• using the enzyme polynucleotide
phosphorylase (catalyzes random synthesis of
RNA chains)
i.e. nNDPs
polynucleotide phosphorylase
(NMP)n + nPi
Using this system they made a polyU mRNA by
programming their reaction with UDP
When this was put into the cell-free extract it
should be translated into a protein made up of
amino acids coded by the codon UUU
Deciphering the first word
(continued)
Experiment:
• They set up 20 different test tube reactions
• Each one was spiked with a different
radioactive amino acid
• They programmed each with the polyU RNA
• Then recovered the proteins by acid
precipitation
• Under these conditions the proteins precipitate
but the free amino acids do not
• Then they asked which reaction (out of the 20)
has radioactivity in the protein pellet?
Biochemists Break the Code
Results
• Marshall Nirenberg and Heinrich Matthaei showed
that poly-U produced polyphenylalanine in a cellfree solution from E. coli. In other words, only the
test tube reaction spiked with radioactive Phe
generated a radioactive pellet
They repeated the experiment with other synthetic
homopolymer RNAs
• Poly-A gave polylysine (AAA = Lys)
• Poly-C gave polyproline (CCC = Pro)
• Poly-G gave polyglycine (GGG = Gly)
Getting at the Rest of the Code
• Work with nucleotide copolymers (poly (A,C), etc.),
revealed some of the codes
• Gobind Khorana (organic chemist)
• -synthesized DNA composed of alternating
copolymers eg: ACACACACACAC…..
• Then used RNAP to make RNA from the DNA
template eg: UGUGUGUGUGUGU……
• This RNA transcript has two possible alternating
codons: UGU GUG UGU GUG
• In a translation extract you should get a protein with
2 alternating amino acids
Nirenberg and Leder
• Took a cell-free translation extract (ribosomes and
tRNAs charged with their specific amino acid)
• Added a synthetic triplet RNA (a codon) eg UUU
• They found that addition of that simple triplet RNA to
the cell-free extract could stimulate the binding of the
tRNA that recognized that codon to a ribosome
• Since the tRNA is covalently linked to the amino acid
that is coded for by the codon, therefore that amino
acid gets localized to the ribosome
• If they collect the ribosomes from the experiment
they can identify which amino acid was brought to
the ribosome by that triplet codon
Nirenberg and Leder
Phe
Ribosome
•Ternary
complex
•Very large
•Can be
captured
on a filter
AAA
UUU
Triplet RNA
Experiment: for each triplet RNA set up 20 reactions, each one
spiked with a different radioactive amino acid. Ask which
reaction generates radioactivity on the filter. That’s the amino
acid coded for by the triplet codon!
Getting at the Rest of the Code
• Finally Marshall Nirenberg and Philip Leder cracked
the entire code in 1964
• They showed that trinucleotides bound to ribosomes
could direct the binding of specific aminoacyl-tRNAs
(See Figure 31.6)
• By using C-14 labeled amino acids with all the
possible trinucleotide codes, they elucidated all 64
correspondences in the code
• Found that all the codons (except the 3 stop codons)
specified an amino acid
• There are 64 codons and 20 amino acids
• Therefore amino acids can be encoded by >1 codon
Features of the Genetic Code
• All the codons have meaning: 61 specify amino
acids, and the other 3 are "nonsense" or "stop"
codons
• The code is unambiguous - only one amino acid is
indicated by each of the 61 codons
• The code is degenerate - except for Trp and Met,
each amino acid is coded by two or more codons
• Codons representing the same or similar amino
acids are similar in sequence
• 2nd base pyrimidine: usually nonpolar amino acid
• 2nd base purine: usually polar or charged aa