Chapter 32

The Genetic CodeIncluding a review of tRNA structure

from Chapter 12 section 7

Review of Steps in Gene Expression

From Access Excellence: http://www.accessexcellence.org/AB/GG/steps_to_Prot.html

RNA = nucleotide sequence

protein = amino acid sequence

Adaptor molecule

Translating the Message

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 #1The 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 #2The 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 #3Crick’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

From Lehninger “Principles of Biochemistry” p 1021

•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

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 (), dihydrouridine (D), ribothymidine (T), and methylated bases (mG, mI)

Unusual bases in tRNAs

Invariant basesAmino acid addition site

Varies in size

Interacts with the ribosome

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 Non-canonical base pairs stabilize the 3o structure

Animation of tRNA secondary and tertiary structure

Elucidation of the Genetic Code4 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

gene

protein

Mutant 1 Mutant 2Mutant 3

Therefore gene sequence is colinear with protein sequence.

What is the nature of the Code

• 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

mRNA (nucleotides)

protein (amino acids)

4 different nucleotides

20 different amino acids

What is the nature of the Code

• 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

X

Changes 3 amino acids

XChanges 1 amino acid

Changes all following amino acids

following amino acids are unchanged

The Nature of the Genetic Codesummary 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

They generated RNAs that are homopolymers • using the enzyme polynucleotide

phosphorylase (catalyzes random synthesis of RNA chains)

Deciphering the first word

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

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?

Deciphering the first word (continued)

Biochemists Break the Code

Results • Marshall Nirenberg and Heinrich Matthaei showed that

poly-U produced polyphenylalanine in a cell-free 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

UUUAAA

Phe

Ribosome•Ternary complex

•Very large

•Can be captured on a filter

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!

Triplet RNA

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