Translation

Protein biosynthesis
Review of Steps in Gene Expression

RNA = nucleotide sequence

Adaptor molecule

protein = amino acid sequence

 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 #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
tRNA
• Activated amino acids are called aminoacyl- tRNAs
• The enzymes that do the activation are called
aminoacyl-tRNA synthetases
 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
•These adaptors 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

 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
The Nature of the Genetic Code

• 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)
 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 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
C Third-Base Degeneracy
C 2nd base pyrimidine: usually nonpolar amino acid
C 2nd base purine: usually polar or charged aa
 Third-Base Degeneracy and The
Wobble Hypothesis
• The first two bases of the codon make
normal H-bond pairs with the 2nd and 3rd
bases of the anticodon
• At the remaining position, non-canonical
pairing may occur
• The rules:
Anticodon Codon
(base #1) (base #3)
C G
A U
G C,U
U A,G
I U,C,A
 tRNA
• tRNAs are the “adaptors” in protein
synthesis
 Review of tRNA Structure
• 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 (ψ ), dihydrouridine (D),
ribothymidine (T), and methylated bases (mG, mI)
 Amino acid
addition site

Interacts with
the ribosome
riant bases

Varies in size

Base pairs with the codon

in the mRNA transcript
tRNA tertiary structure: L-shape
 tRNAs are bifunctional

specific amino acid

•Amino acids must be
Phe
activated for translation
Acceptor stem
•Via covalent linkage of
an amino acid to the
3’OH of the tRNA

Anticodon loop •This generates a

AAA
UUU “charged tRNA”,
Codon in mRNA aminoacyl-tRNA
•
tRNA activation must be specific
•The delivery of the amino acid is specified by this
codon-anticodon interaction (regardless of which amino
acid is attached to the tRNA)
•
•Each tRNA is matched with its amino acid long before it
reaches the ribosome.
•
•The match is made by a collection of remarkable
enzymes, the aminoacyl-tRNA synthetases.
•
•These enzymes charge each tRNA with the proper
amino acid, thus allowing each tRNA to make the proper
translation from the genetic code of DNA into the amino
acid code of proteins.
The Aminoacyl-tRNA Synthetase
Reaction
• The goal of this reaction is to activate an amino
acid by forming an ester linkage with the correct
tRNA

CC O-P-OCH2 Adenine CCAOH 3’

-
O O
acceptor stem

OH OH
O

C
H
- C - R group Amino acid
NH3+
 The Aminoacyl-tRNA Synthetase
Reaction is two steps

1) Activate the amino acid first, by reacting with ATP

Amino acid + ATP Aminoacyl AMP + PPi 2Pi

An enzyme-bound intermediate

2) Transfer the activated amino acid to its cognate

tRNA
Aminoacyl AMP +tRNA Aminoacyl-tRNA + AMP
Aminoacyl-tRNA Synthetases
• All have a common 2-domain structure

A catalytic domain A variable domain

Interacts with the Interacts with the

tRNA 3’OH specific bases on the
tRNA that identify
Recognizes and that tRNA
binds the cognate
amino acid
 Aminoacyl-tRNA Synthetases
High fidelity in translation
• Aminoacyl-tRNA synthetases must perform their
tasks with high accuracy, since every mistake will
result in a misplaced amino acid when new proteins
are constructed.
• These enzymes make about one mistake in 10,000.
• This is a two different levels:
1) They must be able to recognize and bind to the
correct tRNA
2) They must be able to recognize and bind to the
correct amino acid