Molecular Biochemistry II

tRNA & Ribosomes

Copyright 1999-2008 by Joyce J. Diwan.

All rights reserved.

Molecular Biology
Familiarity with basic concepts is assumed, including:
nature of the genetic code
maintenance of genes through DNA replication
transcription of information from DNA to mRNA
translation of mRNA into protein.

DNA

mRNA

protein

Purines & Pyrimidines

Nucleic acids are polymers of nucleotides.
Each nucleotide includes a base that is either
a purine (adenine or guanine), or
a pyrimidine (cytosine, uracil, or thymine).
Nucleoside bases found in RNA:
O

NH2
N

N
N

N
H

adenine (A)

HN
H2N

N
guanine (G)

NH2

N
H

NH

N
N
H
cytosine (C)

N
H
uracil (U)

Some nucleic acids contain modified bases. Examples:

Nucleoside bases found in RNA:
O

NH2
N

N
N

N
H

HN
H2N

adenine (A)

NH2

N
H

NH

N
N
H

guanine (G)

N
H

cytosine (C)

uracil (U)

Examples of modified bases found in tRNA:

O

NH2
H3C

N
H

NH2

+N

CH3
N

HN
H2N

N
H

O
+

CH3

N
N
H

HN
O

NH
N
H

1-methyladenine (m1A) 7-methylguanine (m7G) 3-methylcytosine (m3C) pseudouracil ()

In a nucleotide, e.g., adenosine monophosphate (AMP),

the base is bonded to a ribose sugar, which has a phosphate
in ester linkage to the 5' hydroxyl.
NH2

NH2
N

N
N

N
H

ribose

O3P

5' CH2

O
3'

OH

H
OH

2'

adenosine

N
O

CH2

H 1'

4'

adenine

adenine

HO

NH2

OH

H
OH

adenosine monophosphate (AMP)

Nucleic acids
have a backbone
of alternating Pi &
ribose moieties.
Phosphodiester
linkages form as
the 5' phosphate of
one nucleotide
forms an ester link
with the 3' OH of
the adjacent
nucleotide.
A short stretch of
RNA is shown.

NH2

adenine
N

N
5' end O

O P

NH2

N
5'

CH2

4'

H 1'
H
OH

O
P

5'

CH2

H
H

nucleic acid

ribose

2'

3'

cytosine

H
H
OH

3'

O
P
O

ribose

(etc)
3' end

cytosine (C)

N
N

guanine (G)

N
N
N

N
N
H

NH

G C base
pair in tRNA

Hydrogen bonds link 2 complementary nucleotide bases

on separate nucleic acid strands, or on complementary
portions of the same strand.
Conventional base pairs: A & U (or T); C & G.
In the diagram at left, H-bonds are in red. Bond lengths
are inexact. The image at right is based on X-ray
crystallography of tRNAGln. H atoms are not shown.

Secondary structure
Base pairing over extended stretches of complementary
base sequences in two nucleic acid strands stabilizes
secondary structure, such as the double helix of DNA.
Stacking interactions between adjacent hydrophobic
bases contribute to stabilization of such secondary
structures. Each base interacts with its neighbors above
and below, in the ladder-like arrangement of base pairs
in the double helix, e.g., of DNA.

Genetic code
The genetic code is based on the sequence of bases along
a nucleic acid.
Each codon, a sequence of 3 bases in mRNA, codes for a
particular amino acid, or for chain termination.
Some amino acids are specified by 2 or more codons.
Synonyms (multiple codons for the same amino acid) in
most cases differ only in the 3rd base. Similar codons tend
to code for similar amino acids. Thus effects of mutation
are minimized.

Genetic Code
1st base
U
U
UUU Phe
UUC Phe
UUA Leu
UUG Leu
C
CUU Leu
CUC Leu
CUA Leu
CUG Leu
A
AUU Ile
AUC Ile
AUA Ile
AUG Met*
G
GUU Val
GUC Val
GUA Val
GUG Val
*Met and initiation.

2nd base
C
UCU Ser
UCC Ser
UCA Ser
UCG Ser
CCU Pro
CCC Pro
CCA Pro
CCG Pro
ACU Thr
ACC Thr
ACA Thr
ACG Thr
GCU Ala
GCC Ala
GCA Ala
GCG Ala

A
UAU Tyr
UAC Tyr
UAA Stop
UAG Stop
CAU His
CAC His
CAA Gln
CAG Gln
AAU Asn
AAC Asn
AAA Lys
AAG Lys
GAU Asp
GAC Asp
GAA Glu
GAG Glu

3rd base
G
UGU Cys
UGC Cys
UGA Stop
UGG Trp
CGU Arg
CGC Arg
CGA Arg
CGG Arg
AGU Ser
AGC Ser
AGA Arg
AGG Arg
GGU Gly
GGC Gly
GGA Gly
GGG Gly

U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G

tRNA
The genetic code is read during translation via adapter
molecules, tRNAs, that have 3-base anticodons
complementary to codons in mRNA.
"Wobble" during reading of the mRNA allows some
tRNAs to read multiple codons that differ only in the
3rd base.
There are 61 codons specifying 20 amino acids.
Minimally 31 tRNAs are required for translation, not
counting the tRNA that codes for chain initiation.
Mammalian cells produce more than 150 tRNAs.

RNA structure:
Most RNA molecules have
secondary structure,
consisting of stem & loop
domains.

A
:
U

U
:
A

A
:
U

C
:
G

C UG
C
:
U
G
U
C U

stem

loop

Double helical stem domains arise from base pairing

between complementary stretches of bases within the
same strand.
These stem structures are stabilized by stacking
interactions as well as base pairing, as in DNA.
Loop domains occur where lack of complementarity
or the presence of modified bases prevents base pairing.

anticodon loop

tRNA

acceptor
stem

The cloverleaf model of tRNA emphasizes the two

major types of secondary structure, stems & loops.
tRNAs typically include many modified bases,
particularly in loop domains.

RNA tertiary structure depends on interactions of

bases at distant sites.
These interactions generally involve non-standard
base pairing and/or interactions involving three or
more bases.
Unpaired adenosines (not involved in conventional
base pairing) predominate in participating in nonstandard interactions that stabilize tertiary RNA
structures.
tRNAs have an L-shaped tertiary structure.

anticodon loop

tRNA

Extending from the acceptor stem,

the 3' end of each tRNA has the
sequence CCA.

acceptor
stem

The appropriate amino

acid is attached to the
ribose of the terminal
adenosine (A, in red) at
the 3' end.
The anticodon loop is
at the opposite end of
the L shape.

anticodon

tRNA

Phe

acceptor
stem

#46
(m7G)
#22
G

Tertiary base
Phe
pairs in tRNA

#13
C

#46
(m7G)
#22
G

#13
C

Tertiary base
Phe
pairs in tRNA

An example of non-standard H bond interactions that help

to stabilize the L-shaped tertiary structure of a tRNA, in ball
& stick & spacefill displays.
H atoms are not shown. (From NDB file 1TN2).

Some other RNAs,

including viral RNAs &
segments of ribosomal
RNA, fold in
pseudoknots, tertiary
structures that mimic the
3D structure of tRNA.
Pseudoknots are
similarly stabilized by
non-standard H-bond
interactions.
Explore tRNAPhe with
Chime (PDB file 1TRA).

anticodon

Phe

tRNA

acceptor
stem

H
C

O
C

O
O

Amino acid

NH3+

H
C

CH2

ATP

Adenine

OH

H
OH

O
O

NH2

Aminoacyl-AMP

CH2

O
H

Adenine

OH

H
OH

PPi

Aminoacyl-tRNA Synthetases catalyze linkage of the

appropriate amino acid to each tRNA.
The reaction occurs in 2 steps.
In step 1, an O atom of the amino acid -carboxyl attacks
the P atom of the initial phosphate of ATP.

H
C

O
C

O
O

CH2

NH2

Aminoacyl-AMP

In step 2, the
2' or 3' OH of
the terminal
adenosine of
tRNA attacks
the amino acid
carbonyl C
atom.

OH

H
OH

tRNA
AMP

tRNA

Adenine

O
O

CH2

H
H

Adenine
H
H
OH

O
C

HC

NH3+

(terminal 3nucleotide
of appropriate tRNA)
Aminoacyl-tRNA

Aminoacyl-tRNA Synthetase - summary:

1. amino acid + ATP aminoacyl-AMP + PPi
2. aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP
The 2-step reaction is spontaneous overall, because the
concentration of PPi is kept low by its hydrolysis,
catalyzed by Pyrophosphatase.

There is generally a different Aminoacyl-tRNA Synthetase

(aaRS) for each amino acid.
Accurate translation of the genetic code depends on
attachment of each amino acid to an appropriate tRNA.
Each aaRS recognizes its particular amino acid & tRNAs
coding for that amino acid.
Identity elements: tRNA domains recognized by an aaRS.
Most identity elements are in the
acceptor stem & anticodon loop.

anticodon loop

Aminoacyl-tRNA Synthetases arose

early in evolution.
Early aaRSs probably recognized
tRNAs only by their acceptor stems.

tRNA

acceptor
stem

tRNA
O
O

(terminal 3nucleotide
of appropriate tRNA)
CH2

There are 2 families

of Aminoacyl-tRNA
Synthetases:
Class I & Class II.

H
H

Adenine
H
H
OH

HC

NH3+

Aminoacyl-tRNA

Two different ancestral proteins evolved into the 2 classes

of aaRS enzymes, which differ in the architecture of their
active site domains.
They bind to opposite sides of the tRNA acceptor stem,
aminoacylating a different OH of the tRNA (2' or 3').

Class I aaRSs:
Identity elements usually include residues of the
anticodon loop & acceptor stem.
Class I aaRSs aminoacylate the 2'-OH of adenosine at
their 3' end.
Class II aaRSs:
Identity elements for some Class II enzymes do not
include the anticodon domain.
Class II aaRSs tend to aminoacylate the 3'-OH of
adenosine at their 3' end.

Proofreading/quality control:
Some Aminoacyl-tRNA Synthetases are known to have
separate catalytic sites that release by hydrolysis
inappropriate amino acids that are misacylated or mistransferred to tRNA.
E.g., the aa-tRNA Synthetase for isoleucine (IleRS) a small
percentage of the time activates the closely related amino
acid valine to valine-AMP.
After valine is transferred to tRNAIle, to form Val-tRNAIle, it
is removed by hydrolysis at a separate active site of IleRS
that accommodates Val but not the larger Ile.
In some bacteria, editing of some misacylated tRNAs is
carried out by separate proteins that may be evolutionary
precursors to editing domains of aa-tRNA Synthetases.

Some amino acids are modified after being linked to a

tRNA. Examples:
In prokaryotes the initiator tRNAfMet is first charged with
methionine.
Methionyl-tRNA formyltransferase then catalyzes
formylation of the methionine, using tetrahydrofolate
as formyl donor, to yield formylmethionyl-tRNAfMet.
In some prokaryotes, a non-discriminating aaRS
loads aspartate onto tRNAAsn.
The aspartate moiety is then converted by an amidotransferase to asparagine, yielding Asn-tRNAAsn.
Glu-tRNAGln is similarly formed and converted to GlntRNAGln in such organisms.

 Some proteins contain the

unusual amino acid
selenocysteine (Sec), with
selenium substituting for the S
atom in cysteine.

There is a selenocysteine tRNA that differs from other

tRNAs, e.g., in having a slightly longer acceptor stem & a
unique modified base in the anticodon loop.
tRNASec is loaded with serine via Seryl-tRNA Synthetase.
The serine moiety is then converted to selenocysteine by
another enzyme, in a reaction involving selenophosphate.
Sec-tRNASec utilization during protein synthesis requires
special elongation factors because the codon for
selenocysteine is UGA, which normally is a stop codon.

Other roles of aminoacyl-tRNA synthetases:

In some organisms, Aminoacyl-tRNA Synthetases
(aaRSs) have evolved to take on signaling roles in
addition to the catalytic role of joining an amino acid
to the correct tRNA.
Examples have been identified of particular aaRSs
that regulate transcription, translation or intron
splicing through binding to DNA or RNA.

 Proteolytic cleavage of the human aaRSTyr yields a

cytokine that stimulates angiogenesis.
A truncated form of the human aaRSTrp inhibits
angiogenesis.
Regulation of apoptosis by the human aaRSGln is
dependent on the concentration of its substrate
glutamine.
Several mammalian Aminoacyl-tRNA Synthetases
associate with other proteins to form large
macromolecular complexes whose roles are actively
being investigated.

Ribosome Composition (S = sedimentation coefficient)

Ribosome
Source
E. coli

Whole
Ribosome
70S

Small
Subunit
30S
16S RNA
21 proteins

Rat
cytoplasm

80S

40S
18S RNA
33 proteins

Large
Subunit
50S
23S & 5S
RNAs
31 proteins
60S
28S, 5.8S, &5S
RNAs
49 proteins

Eukaryotic cytoplasmic ribosomes are larger and more

complex than prokaryotic ribosomes. Mitochondrial and
chloroplast ribosomes differ from both examples shown.

5S rRNA
crown view
displayed as
ribbons & sticks.

PDB 1FFK

Structures of large & small subunits of bacterial &

eukaryotic ribosomes have been determined, by X-ray
crystallography & by cryo-EM with image reconstruction.
Consistent with predicted base pairing, X-ray crystal
structures indicate that ribosomal RNAs (rRNAs) have
extensive secondary structure.

Structure of the E. coli Ribosome

large subunit

tRNA

EF-G

small subunit

mRNA
location

The cutaway view at right shows positions of tRNA (P, E

sites) & mRNA (as orange beads). EF-G will be discussed
later. This figure was provided by Joachim Frank, whose
lab at the Wadsworth Center carried out the cryo-EM and
3D image reconstruction on which the images are based.

Small Ribosomal Subunit

In the translation complex, mRNA threads through a
tunnel in the small ribosomal subunit.
tRNA binding sites are in a cleft in the small subunit.
The 3' end of the 16S rRNA of the bacterial small
subunit is involved in mRNA binding.
The small ribosomal subunit is relatively flexible,
assuming different conformations.
E.g., the 30S subunit of a bacterial ribosome was
found to undergo specific conformational changes
when interacting with a translation initiation factor.

30S ribosomal subunit

PDB 1FJF

Small
ribosomal
subunit of a
thermophilic
bacterium:
rRNA in
monochrome;
proteins in
spacefill display
ribbons
varied colors.
The overall shape of the 30S ribosomal subunit is largely
determined by the rRNA. The rRNA mainly consists of
double helices (stems) connected by single-stranded loops.
The proteins generally have globular domains, as well as
long extensions that interact with rRNA and may stabilize
interactions between RNA helices.

Large ribosome subunit:

The interior of the large
subunit is mostly RNA.
Proteins are distributed
mainly on the surface.

PDB 1FFK

Large
Ribosome
Subunit

Some proteins have long

tails that extend into the
interior of the complex.
These tails, which are
highly basic, interact
with the negatively
charged RNA.

"Crown" view with RNAs blue, in

spacefill; proteins red, as backbone.

PDB 1FFK

The active site domain

for peptide bond
formation is essentially
devoid of protein.

Large
Ribosome
Subunit

The peptidyl transferase

function is attributed to
23S rRNA, making this
RNA a "ribozyme."
"Crown" view with RNAs blue, in
spacefill; proteins red, as backbone.

Protein synthesis
takes place in a cavity
within the ribosome,
between small & large
subunits.

PDB 1FFK

Nascent polypeptides
emerge through a
tunnel in the large
subunit.
The tunnel lumen is
lined with rRNA
helices and some
ribosomal proteins.

Large ribosome subunit.

Backbone display with RNAs blue. View
from bottom at tunnel exit.

Catalysis of protein synthesis and movement of the

ribosome relative to messenger RNA are accompanied by
changes in ribosome conformation.
EM & X-ray crystallographic studies, carried out in the
presence & absence of initiation & elongation factors as
well as inhibitors of protein synthesis, have revealed
conformational changes in rRNA.
Thus rRNA participates in conformational coupling in
addition to its structural & catalytic roles.
tRNAs also undergo substantial conformational changes
within ribosomal binding sites during protein synthesis.
Explore the large ribosomal subunit.