Section P
Genetic code and tRNA
P1 The genetic code
P2 tRNA structure and function
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
P1 The genetic code
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Nature
Deciphering
Feature
Effect of Mutation
Universality
ORFs
Overlapping Genes
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Nature
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Genetic code is a triplet code (to be grouped in 3nt);
The triplet codons are adjacent (non-overlapping);
They are not separated by punctuation (comma-less).
Because many of the 64 codons specify the same
amino acid, the genetic code is degenerate;
• So that the genetic codons have redundancy.
As more gene and protein sequence information has been
obtained, it has become clear that the genetic code is
very nearly, but not quite, universal. This supports the
hypothesis that all life has evolved from a single
common origin.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Deciphering
• In the 1960s, Marshall Nirenberg developed a “cell-free
protein synthesizing system” from E. coli (细胞外蛋白合成体系).
• To determine which amino acids were being polymerized into
polypeptides, it was necessary nucleotide phosphorylase was used
to make synthetic mRNAs that were composed of only one
nucleotide, that is poly (U), poly (C), poly (A) and poly (G).
• If protein synthesis took place after adding one of these mRNAs,
then in one of the 20 reaction tubes, the radioactivity could show
the formation of polypeptide.
• In this way, it was found that:
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Poly (U) caused the synthesis of poly-phonylalanine,
Poly (C) coded for poly-proline
Poly (A) for poly-lysine
Poly (G) did not work because it formed a complex secondary structure.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Deciphering
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Deciphering
• The precise sequence of the triplet
codon can only be worked out if
additional information is available.
• Towards the end of the 1960s, it was
found that synthetic tri-nucleotides
could attach to the ribosome
and bind their corresponding
aminoacyl-tRNAs.
• Upon filtering through a membrane,
only the complex of ribosome, synthetic
triplet and aminoacyl-tRNA was
retained on the membrance.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Feature
• The genetic code is degenerate (or it
shows redundancy). This is because 18
out of 20 amino acids have more than one
codon to specify them, called
synonymous codons .
• Only methionine and tryptophan have
single codons.
• The synonymous codons are not
positioned randomly, but are grouped in
the table. Generally they differ only in
their third position.
• In all cases, if the third position is a
pyrimidine, then the codons specify the
same amino acid (are synonymous).
• In most cases, if the third position is a
purine the codons are also synonymous.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Universality
• For a long time after the
genetic code was deciphered,
it was thought to be universal,
that is the same in all
organisms.
• However, since 1980, it has
been discovered that
mitochondria, which have their
own small genomes, use a
genetic code that differs
slightly from the standard, or
‘universal’ code. Indeed, it is
now known that some other
unicellular organisms also
have a variant genetic code.
Section O: RNA Processing and RNPs.
P254, P1.2
Modifications of the genetic code
Codon Usual Alternative Organelle/organism
AGA
AGG
Some Animal
Arg Stop, Ser
Mitochondria
AUA
Ile
CGG
Arg Trp
Plant Mitochondria
CUN
Leu Thr
Yeast Mitochondria
AUU
GUG
UUG
UAA
UAG
Ile
Val Start
Leu
Some Prokaryotes
Stop Glu
Some Protozoans
UGA
Stop Trp
Mit. Mycoplasma
Met
Mitochondria
Yang Xu, College of Life Sciences
Overlapping Genes
• Generally overlapping genes occur where the genome size is
small and there is a need for greater information storage density.
• In viruses, for example, the phage ΦΧ174 makes 11 proteins of
combined molecular mass 262 kDa from a 5386 bp genome.
Without overlapping genes, this genome could encode at most
200 kDa of protein. Three proteins are encoded within the
coding regions for longer proteins.
• In prokaryotes, the ribosomes simply have to find the second
start codon to be able to translate the overlapping gene and they
may achieve this without detaching from the template.
• Eukaryotes have a different way of initiating protein synthesis
and tend to make use of alternative RNA processing to generate
variant proteins from one gene.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
P2 tRNA Structure
and Function
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tRNA Primary Structure
tRNA Secondary Structure
tRNA Tertiary Structure
tRNA Function
Aminoacylation of tRNA
Aminoacyl-tRNA Synthetases
Proofreading
Section O: RNA Processing and RNPs.
Robert Holley(46y)
Yang Xu, College of Life Sciences
tRNA Primary Structure
• tRNAs are the adaptor molecules that deliver ammo acids to the
ribosome and decode the information in mRNA. Their primary
structure (i.e. the linear sequence of nucleotides) is 60-95 nt long,
but most commonly 76 nt.
• They have many modified bases sometimes accounting for 20%
of the total bases in any one tRNA molecule. Indeed, over 50
different types of modified base have been observed in the
several hundred tRNA molecules characterized to date, and all of
them are created post-transcriptionally.
• Seven of the most common types are shown in Fig. 1 as
nucleosides.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
tRNA Secondary Structure
• All tRNAs have a common secondary structure (i.e. base
pairing of different regions to form stems and loops), the
cloverleaf structure.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
---aa accept arm
loading aa at 3’ end
---TΨC loop:
contact with 5s rRNA
---DHU loop:
contact with AARS
---anti-codon loop:
34
34th is wobble base
Section O: RNA Processing and RNPs.
---extra loop: (Variable arm)
classification
marker
? Sciences
Yang Xu, College
of Life
tRNA Tertiary Structure
• There are hydrogen bonds (tertiary hydrogen bonds) that
help form the 3-D structure of tRNA molecules.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
tRNA Function
• tRNAs are joined to amino adds to become aminoacyl-tRNAs
(charged tRNAs) in a reaction called aminoacylation.
• It is these charged tRNAs that are the adaptor molecules in
protein synthesis.
• Special enzymes called aminoacyl-tRNA synthetases carry out
the joining reaction which is extremely specific (i.e. a specific
amino acid is joined to a specific tRNA).
• These pairs of specific amino acids and tRNAs, or tRNAs and
aminoacyl-tRNA synthetases are called cognate pairs, and the
nomenclature used is shown in Table 1 (page 258).
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Aminoacylation of tRNA
• The general aminoacylation reaction is shown in Fig. It is a
two-step reaction driven by ATP:
• In the first step, AMP is linked to the carboxyl group of the
amino acid giving a high-energy intermediate called an
aminoacyl adenylate.
• The hydrolysis of the pyro-phosphate released (to two
molecules of inorganic phosphate) drives the reaction forward.
• In the second step, the aminoacyl adenylate reacts with the
appropriate uncharged tRNA to give the aminoacyl-tRNA and
AMP.
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Aminoacyl-tRNA
Step 1
Setp 2
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Aminoacyl-tRNA Synthetases
• Despite the fact that they all carry out the same reaction of
joining an amino acid to a tRNA, the various synthetase
enzymes can be quite different.
• They fall into one of four classes of subunit structure, being
either a, a2, a4, a2b2.
• The polypeptide chains range from 334 to over 1000 amino
acids in length, and these enzymes contact the tRNA on the
underside (in the angle) of the L-shape.
• They have a separate amino acid-binding site. The synthetases
have to be able to distinguish between about 40 similarly
shaped, but different, tRNA molecules in cells, and they use
particular parts of the tRNA molecules, called identity
elements, to be able to do this (Fig. 4).
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
Identity Elements (Paracodon)
AARS
tRNA binding site
Paracodon of tRNA
Section O: RNA Processing and RNPs.
aa binding site
loading
Amino Acid（R）
Yang Xu, College of Life Sciences
tRNAPhe
tRNAAla
Identity Elements in
various tRNA Molecules
tRNAfMet
Section O: RNA Processing and RNPs.
Ser
tRNA
Yang Xu, College of Life Sciences
Position of Identity Elements
TΨC loop
aa arm: A,D,G,H,N,S,T,V,W
73th site
A.C.D.E.F.G.H.I.
K.L.M.N.P.Q.R.S
.V.W.Y.
Extra loop
A, F, L, R
Anti-codon loop
C,D,E,F,G,H,I,K,M,N,P
Q,R,T,V,W,Y
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences
That’s all for Section P
Section O: RNA Processing and RNPs.
Yang Xu, College of Life Sciences