-
BackgroundProteins are made from combinations of 20 different
aminoacids. The genes that encode proteinsthat is, specify thetype
and linear order of their component amino acidsarelocated in DNA, a
polymer made up of only four differentnucleotides. The DNA code is
transcribed into RNA, whichis also composed of four nucleotides.
Nirenbergs studieswere premised on the hypothesis that the
nucleotides inRNA form codewords, each of which corresponds to
oneof the amino acids found in protein. During protein syn-thesis,
these codewords are translated into a functional pro-tein. Thus, to
understand how DNA directs protein synthe-sis, Nirenberg set out to
understand the relationshipbetween RNA codewords and protein
synthesis.
At the outset of his studies, much was already knownabout the
process of protein synthesis, which occurs onribosomes. These large
ribonucleoprotein complexes canbind two different types of RNA:
messenger RNA(mRNA), which carries the exact protein-specifying
codefrom DNA to ribosomes, and smaller RNA molecules nowknown as
transfer RNA (tRNA), which deliver aminoacids to ribosomes. tRNAs
exist in two forms: those thatare covalently attached to a single
amino acid, known asamino-acylated or charged tRNAs, and those that
haveno amino acid attached called uncharged tRNAs. Afterbinding of
the mRNA and the amino-acylated tRNA to the
ribosome, a peptide bond forms between the amino acids,beginning
protein synthesis. The nascent protein chain iselongated by the
subsequent binding of additional tRNAsand formation of a peptide
bond between the incomingamino acid and the end of the growing
chain. Althoughthis general process was understood, the
questionremained: How does the mRNA direct protein synthesis?
When attempting to address complex processes such asprotein
synthesis, scientists divide large questions into aseries of
smaller, more easily addressed questions. Prior toNirenbergs study,
it had been shown that when phenyl-alanine-charged tRNA was
incubated with ribosomes andpolyuridylic acid (polyU), peptides
consisting of onlyphenylalanine were produced. This finding
suggested thatthe mRNA codeword, or codon, for phenylalanine is
madeup of the nucleotides containing the base uracil.
Similarstudies with polycytadylic acid (polyC) and polyadenylicacid
(polyA) showed these nucleotides containing thebases cytadine and
adenine made up the codons for prolineand lysine, respectively.
With this knowledge in hand,Nirenberg asked the question: What is
the minimum chainlength required for tRNA binding to ribosomes? The
sys-tem he developed to answer this question would give himthe
means to determine which amino-acylated tRNAwould bind which mRNA
codon, effectively cracking thegenetic code.
Classic Experiment 4.1
Cracking the Genetic Code
By the early 1960s molecular biologists had adopted the
so-called cen-tral dogma, which states that DNA directs synthesis
of RNA (tran-scription), which then directs assembly of proteins
(translation). However,
researchers still did not completely understand how the code
embodied in
DNA and subsequently in RNA directs protein synthesis. To
elucidate this
process, Marshall Nirenberg embarked upon a series of studies
that would
lead to solution of the genetic code.
-
The ExperimentThe first step in determining the minimum length
of mRNArequired for tRNA recognition was to develop an assay
thatwould detect this interaction. Since previous studies hadshown
that ribosomes bind mRNA and tRNA simultane-ously, Nirenberg
reasoned that ribosomes could be used asa bridge between a known
mRNA codon and a knowntRNA. When the three components of protein
synthesis areincubated together in vitro, they should form a
complex.After devising a method to detect this complex,
Nirenbergcould then alter the size of the mRNA to determine
theminimum chain length required for tRNA recognition.
Before he could begin his experiment, Nirenberg need-ed both a
means to separate the complex from unboundcomponents and a method
to detect tRNA binding to theribosome. To isolate the complex he
exploited the abilityof nylon filters to bind large RNA molecules,
such as ribo-somes, but not the smaller tRNA molecules. He used
anylon filter to separate ribosomes (and anything bound tothe
ribosomes) from unbound tRNA. To detect the tRNAbound to the
ribosomes, Nirenberg used tRNA chargedwith amino acids that
contained a radioactive label, C.All other components of the
reaction were not radioactive.Since only ribosome-bound tRNA is
retained by the nylon
14
membrane, all radioactivity found on the nylon
membranecorresponds to tRNA bound to ribosomes. Now, a systemwas in
place to detect the recognition between an mRNAmolecule and the
proper amino-acylated tRNA.
To test his system, Nirenberg used polyU as themRNA, and [
C]-phenylalanine-charged tRNA, whichbinds to ribosomes in the
presence of polyU. Ribosomeswere incubated with both polyU and [
C]-phenylalaninetRNA for sufficient time to allow both molecules to
bindto the ribosomes; the reaction mixtures were then passedthrough
a nylon membrane. When the membranes wereanalyzed using a
scintillation counter, they containedradioactivity, demonstrating
that in this system polyUcould recognize phenylalanine-charged
tRNA. But was thisrecognition specific for the proper
amino-acylated tRNA?As a control, [ C]-lysine and [
C]-proline-charged tRNAsalso were incubated with polyU and
ribosomes. After thereaction mixtures were passed through a nylon
filter, noradioactivity was detected on the filter. Therefore,
theassay measured only specific binding between an mRNAand its
corresponding amino-acylated tRNA.
Now, the minimum chain length of RNA necessary forproper
amino-acylated tRNA recognition could be deter-mined. Short
oligonucleotides were tested for their abilityto bind ribosomes and
recognize the appropriate tRNA.
1414
14
14
-
When UUU, a trinucleotide, was used, tRNA binding toribosomes
could be detected. However, when the UU di-nucleotides was used, no
binding was detected. This resultsuggested that the codon required
for proper recognitionof tRNA is a trinucleotide. Nirenberg
repeated this exper-iment on two other homogeneous trinucleotides,
CCC andAAA. When these trinucleotides were independentlybound to
ribosomes, CCC specifically recognized proline-charged tRNA and AAA
recognized lysine-chargedtRNAs. Since none of the three homogeneous
trinu-cleotides recognized other charged tRNAs, Nirenberg
con-cluded that trinucleotides could effectively direct the prop-er
recognition of amino-acylated tRNAs.
This study accomplished much more than determiningthe length of
the codon required for proper tRNA recogni-tion. Nirenberg realized
that his assay could be used to testall 64 possible combinations of
trinucleotides (see Figure4.1). A method for cracking the code was
available!
DiscussionCombined with the technology to generate
trinucleotidesof known sequence, Nirenbergs assay provided a way
toassign each specific amino acid to one or more
specifictrinucleotides. Within a few years, the genetic code
wascracked, all 20 amino acids were assigned at least one
tri-nucleotide, and 61 of the 64 trinucleotides were found
tocorrespond to an amino acid. The final three trinu-cleotides, now
known as stop codons, signal termina-tion of protein synthesis.
With the genetic code cracked, biologists could readthe gene in
the same manner that the cell did. Simply byknowing the DNA
sequence of a gene, scientists can nowpredict the amino acid
sequence of the protein it encodes.For his innovative work,
Nirenberg was awarded theNobel Prize for Physiology and Medicine in
1968.
PheTrinucleotide and all tRN Aspass through filter
Trinucleotide
A minoacyl-tRN As
Riboso m es stick to filter
Riboso m es
Co m plex of riboso m e , U U U ,and Phe-tRN A sticks to
filter
U U U
U U U
U U U
U U U
Phe
Phe
Leu
Leu
Leu
Arg Arg
ArgArg
Phe
Leu
Figure 4.1Assay deve loped by Marshall N irenberg and his
collaborators for deciphering the genetic code . They prepared 20 E
. coli extracts contain-ing all the am inoacyl-tRNAs (tRNAs w ith
am ino acid attached). In each extract sample , a different am ino
acid was radioactive ly labe led(green); the other 19 am ino acids
w ere present on tRNAs but remained unlabe led. Am inoacyl-tRNAs
and trinucleotides passed through anylon filter w ithout binding
(left pane l); ribosomes, how ever, bind to the filter (center pane
l). Each of the 64 possible trinucleotides wastested separate ly
for its ability to attract a specific tRNA by adding it w ith
ribosomes to different extract samples. Each sample was
thenfiltered. If the added trinucleotide causes the radiolabe led
am inoacyl-tRNA to bind to the ribosome , then radioactivity is
detected on thefilter; otherw ise , the labe l passes through the
filter (right pane l). By synthesizing and testing all possible
trinucleotides, the researchersw ere able to match all 20 am ino
acids w ith one or more codons (e .g., phenylalanine w ith UUU as
shown here). [From H . Lodish et al.,1995, Molecular Ce ll B
iology, 3rd ed. W . H . Freeman and Company. See M . W . N irenberg
and P. Leder, 1964, Science 145:1399].
