Modified from http://www.mhhe.com/brooker
BIO 184
Fall 2006
LECTURE 3
Lecture 3:
Translation of mRNA
Signals for Recognition of mRNA by Ribosomes in Prokaryotes and Eukaryotes. In prokaryotes,
the Shine-Delgarno sequence is recognized and base-paired by the 16S ribosomal RNA in the
small ribosomal subunit. This places the start codon (AUG) in the right place within the ribosome
for translation to begin. In eukaryotes, the small ribosomal subunit recognizes the 5’ cap on the
mRNA. (This is actually mediated by cap binding proteins - not shown). The ribosome then scans
down the mRNA and finds the correct start codon for that gene.
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.figgrp.1177
I. Overview of Translation
The translation of the mRNA codons into amino acid
sequences leads to the synthesis of poypeptides, which then
fold and/or aggregate to form functional molecules called
proteins.
The word “translation” is well-chosen because the chemical
language of nucleic acids (in mRNA) is being changed into
the chemical language of polypeptides during the process.
Page 1
Modified from http://www.mhhe.com/brooker
BIO 184
Fall 2006
LECTURE 3
Proteins are the active participants in cell structure and function.
 They are the “work horses” of the cell
 The main function of the genetic material is, therefore, to encode the
production of cellular proteins in the correct cell, at the proper time, and in
suitable amounts
II. Early Experiments
A. Archibald Garrod (early 20th Century)




Was the first to propose a relationship between genes and protein
production
Garrod studied patients who had defects in their ability to metabolize
certain compounds
He was particularly interested in alkaptonuria
o Patients bodies accumulate abnormal levels of homogentisic acid
(alkapton)
o Disease characterized by black urine and bluish-black discoloration of
cartilage and skin
He proposed that alkaptonuria was due to a missing enzyme, namely
homogentisic acid oxidase

Garrod also knew that alkaptonuria follows a recessive pattern of
inheritance …

He thus proposed that a relationship exists between the inheritance of
the trait and the inheritance of a defective enzyme
o He described the disease as an “inborn error of metabolism”
B. Beedle and Tatum (early 1940s)

George Beadle and Edward Tatum were also interested in the relationship
among genes, enzymes and traits
o They specifically wanted to know whether each gene coded for a
single enzyme or whether each gene coded for many enzymes

Their genetic model was Neurospora crassa (a common bread mold)
o Their studies involved the analysis of simple nutritional requirements
Page 2
Modified from http://www.mhhe.com/brooker
BIO 184
Fall 2006
LECTURE 3

They analyzed more than 2,000 strains that had been irradiated to produce
mutations

They found three strains that were unable to grow on minimal medium.
However, in each case, growth was restored if only a single vitamin is added
to the minimal medium
 1st strain  Pyridoxine
 2nd strain  Thiamine
 3rd strain  p-aminobenzoic acid

In the normal strains, these vitamins were synthesized by cellular enzymes
o In the mutant strains, a genetic defect in one gene prevented the
synthesis of one protein required to produce that vitamin
o Beadle and Tatum’s concluded that a single gene controlled the
synthesis of a single enzyme
 This was referred to as the “one gene–one enzyme theory”

In later decades, this theory had to be modified in two ways:
1. Enzymes are only one category of proteins
2. Some proteins are composed of two or more different polypeptides
 The term polypeptide denotes structure
 The term protein denotes function
For example:
Adult hemoglobin is coded by two genes:
alpha-globin and beta-globin. Two alphaglobin polypeptides (AKA subunits) and
two beta-globin polypeptides must fold
properly and then bind together to make
functional hemoglobin.
Page 3
Modified from http://www.mhhe.com/brooker
BIO 184
Fall 2006
LECTURE 3
B. Khorana (early 1960s)
The Genetic Code, which links the sequence of nucleotides in an mRNA to the
sequence of amino acids in a polypeptide, was deciphered in the early 1960s
 More than one set of experiments were performed
 Only the research performed by a team led by Gobind Khorana will be
described here
Gobind Khorana and his collaborators developed a novel method to synthesize RNA
 They first created short RNAs (2 to 4 nucleotide long) that had a defined
sequence
 These were then linked together enzymatically to create long copolymers
 They used these copolymers in a cell-free translation system to see what
polypeptides were made
o For example, when a copolymer of “UC” was made:
 UCUCUCUCUCUCUCUCUCUC was translated as either serine or
leucine. This meant that UCU and CUC must code for these two
amino acids
Page 4
Modified from http://www.mhhe.com/brooker
BIO 184
Fall 2006
LECTURE 3
III. Characteristics of the Genetic Code
1. It is a Triplet Code

Since life uses 20 amino acids, biologists reasoned that the genetic code
must contain at least 3 letters
o A one-letter code could only encode 4 amino acids
o A two-letter code could only encode 16 amino acids
o A three-letter code could encode 64 amino acids – more than enough



Khorana’s team provided experimental support for a 3-letter code
Each 3-letter combination is called a codon
The code is arbitrary but must have been adopted very early in the evolution
of life since all organisms use almost the same code
2. It has START and STOP codons
Page 5
Modified from http://www.mhhe.com/brooker
BIO 184


Fall 2006
LECTURE 3
AUG (which specifies methionine) = start codon
o AUG also specifies additional methionines within the coding sequence
UAA, UAG and UGA = termination, or stop, codons
2. It is degenerate
 More than one codon can specify the same amino acid
o For example: GGU, GGC, GGA and GGG all code for lysine
 In most instances, the third base is the degenerate base
o It is sometime referred to as the wobble base
3. The code is (nearly) universal
 All living organisms use the same code
o Only a few rare exceptions have been noted, and these are thought to
have evolved after the code was first established
4. All nucleotides are read only once
 Once translation has begun, nucleotides in the mRNA are read in successive
triplets (codons)
o Nucleotides are never skipped or read more than once
The figure below illustrates the process of translation of an mRNA using the
Genetic Code.
Page 6
Modified from http://www.mhhe.com/brooker
BIO 184

Fall 2006
LECTURE 3
Note that the mRNA begins with a 5’ untranslated region
o In other words, the START codon is not at the 5’ end of the mRNA
but somewhat downstream (3’)
o Although it is not shown here, the STOP codon is likewise not at the 3’
end of the mRNA. There is a 3’ untranslated region after it.
IV. Protein Structure
There are four levels of structures in proteins:
1. Primary

Is it’s amino acid sequence

Within the cell, the protein will not be
found in this linear state

Rather, it will adapt a compact 3-D
structure

Indeed, this folding can begin during
translation

The progression from the primary to the
3-D structure is dictated by the amino
acid sequence within the polypeptide
The amino acid
sequence of the
enzyme
lysozyme
129 amino acids long
Page 7
Modified from http://www.mhhe.com/brooker
BIO 184

Fall 2006
LECTURE 3
There are 20 amino acids that may be found in polypeptides
o Each contains a different side chain, or R group
Non-polar amino acids hate
water and are usually found
buried together deep inside
a protein or in regions that
span the plasma membrane
of various organelles or the
cell itself
Polar amino
acids like water
and are often
found on the
surface of
proteins.
Page 8
Modified from http://www.mhhe.com/brooker
BIO 184
Fall 2006
LECTURE 3
2. Secondary

The primary structure of a protein folds to form regular, repeating shapes
known as secondary structures
o There are two types of secondary structures
 a helix
 b sheet
o These are stabilized by the formation of hydrogen bonds between
atoms along the “backbone” of the polypeptide
3. Tertiary

The short regions of secondary structure in a protein fold into a threedimensional tertiary structure
o This is the final conformation of proteins that are composed of a
single polypeptide
o The tertiary structure is stabilized via interactions between R groupd
of the amino acids
 Ionic bonds
 Hydrophic interactions
 Disulfide bonds
 Hydrogen bonds
 Van der Waals forces
4. Quaternary

Proteins made up of two or more polypeptides have a quaternary structure
o This is formed when the various polypeptides associate together to
make a functional protein
o Often stabilized by disulfide bonds
See Figure 13.6, Brooker
V. tRNA Structure and Function
A. The Adaptor Hyothesis
Page 9
Modified from http://www.mhhe.com/brooker
BIO 184
Fall 2006
LECTURE 3
In the 1950s, Francis Crick and Mahon Hoagland proposed the adaptor hypothesis,
which hypothesized that tRNAs play a direct role in the recognition of codons
in the mRNA.
 In particular, the hypothesis proposed that tRNA has two functions:
1. Recognizing a 3-base codon in mRNA
2. Carrying an amino acid that is specific for that codon to the
translation machinery
During mRNA-tRNA recognition, the anticodon in tRNA binds to a complementary
codon in mRNA:
tRNAs are named
according to the
amino acid they bear
Proline
anticodon
B. tRNA Secondary Structure
The secondary structure of tRNAs exhibits a cloverleaf pattern containing:
 Three stem-loop structures:
o Variable region
o An acceptor stem (amino acid binding site)
o 3’ single strand region (anti-codon)
 The actual three-dimensional or tertiary structure involves additional
folding

See Figure 13.10, Brooker
Page 10
Modified from http://www.mhhe.com/brooker
BIO 184
Fall 2006
LECTURE 3
C. Modified Nucleotides and the Wobble Hyothesis
In addition to the normal A, U, G and C nucleotides, tRNAs commonly contain
modified nucleotides
 More than 60 of these can occur
As mentioned earlier, the genetic code is degenerate
 With the exception of serine, arginine and leucine, this degeneracy always
occurs at the codon’s third position
o To explain this pattern of degeneracy, Francis Crick proposed in 1966
the wobble hypothesis
o In the codon-anticodon recognition process, the first two positions
pair strictly according to the A – U /G – C rule
o However, the third position can actually “wobble” or move a bit, thus
tolerating certain types of mismatches
The modified nucleotides in the tRNA anticodon allow this “wobbling” to occur.
VI. The Ribosome
Translation occurs on the surface of a large macromolecular complex termed the
ribosome
A ribosome is composed of structures called the large and small subunits
o Each subunit is formed from the assembly of
 Proteins
 Ribosomal RNA (rRNA)
Ribosomes contain 3 discrete sites:



Peptidyl site (P site)
Aminoacyl site (A site)
Exit site (E site)
Page 11
Modified from http://www.mhhe.com/brooker
BIO 184
Fall 2006
LECTURE 3
Translation can be viewed as occurring in three stages:
1. Initiation
In bacteria:
 The binding of mRNA to the 30S subunit is facilitated by a ribosomalbinding site or Shine-Dalgarno sequence
o This is complementary to a sequence in the 16S rRNA within the small
bacterial ribosomal subunit
16S rRNA
Page 12
Modified from http://www.mhhe.com/brooker
BIO 184
Fall 2006
LECTURE 3
The only charged
tRNA that ever
enters through the
P site
70S initiation
complex
This marks the
end of the first
stage
In eukaryotes:
 Quite different than in bacteria
o Recognition of the mRNA by the ribosome is through proteins (cap
bainding proteins) attached to the 5’ cap of the mRNA
o These are joined by a complex consisting of the 40S subunit,
tRNAmet, and other initiation factors
o The entire assembly moves along the mRNA scanning for the right
start codon
o Once it finds this AUG (usually, but not always, the first one it
encounters), the 40S subunit binds to it
o The 60S subunit then joins, forming the 80S initiation complex
Page 13
Modified from http://www.mhhe.com/brooker
BIO 184
Fall 2006
LECTURE 3
2. Elongation
 During this stage, the amino acids are added to the polypeptide chain, one at
a time
See Figure 13.18, Brooker
3. Termination
 The final stage occurs when a stop codon is reached in the mRNA
o In most species there are three stop or nonsense codons
 UAG
 UAA
 UGA
o These codons are not recognized by tRNAs, but by proteins called
release factors
 Indeed, the 3-D structure of release factors mimics that of
tRNAs
See Figure 13.19, Brooker
VII. The Newly-translated Polypeptide
Polypeptide synthesis has a directionality that parallels the 5’ to 3’ orientation of
mRNA
 During each cycle of elongation, a peptide bond is formed between the last
amino acid in the polypeptide chain and the amino acid being added
Page 14
Modified from http://www.mhhe.com/brooker
BIO 184
Fall 2006
LECTURE 3
VIII. Polypeptide Localization
The amino acid sequences of newly-synthesized polypeptides contain “sorting
signals” that tell the cell where they belong




These are especially important in eukaryotes, where each sorting signal is
recognized by a specific cellular component
These cellular components facilitate the sorting of the protein to its correct
compartment
Many polypeptides will not fold properly and become functional proteins until
they are properly localized within the cell
Sometimes, mutations lead to changes in the amino acid sequence of the
polypeptide that prevent its localization
 Such polypeptides are eventually degraded
 Localization mutations are usually null mutations (the protein coded
by the gene has no residual function in the cell)
Page 15