Transcript Molecular Biochemistry II tRNA & Ribosomes Copyright © 1999-2008 by Joyce J.
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).
N NH 2 N
Nucleoside bases found in RNA:
O NH 2 N HN N O NH N N H H 2 N N N H N H O N H adenine (A) guanine (G) cytosine (C) uracil (U) O H 3 C + N NH 2 N HN O + N CH 3 NH 2 + N CH 3 HN O NH N N H H 2 N N N H N H O N H O 1-methyladenine (m 1 A) 7-methylguanine (m 7 G) 3-methylcytosine (m 3 C) pseudouracil ( )
Some nucleic acids contain modified bases. Examples: N NH 2 N
Nucleoside bases found in RNA:
O NH 2 N HN N O NH H 3 C + N N N H H 2 N N N H N H O N H adenine (A) guanine (G) cytosine (C) uracil (U) O NH 2 N
Examples of modified bases found in tRNA:
O CH 3 NH 2 + N CH 3 HN + N HN O NH N N H H 2 N N N H N H O N H O 1-methyladenine (m 1 A) 7-methylguanine (m 7 G) 3-methylcytosine (m 3 C) 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.
N NH 2 N N NH 2 adenine N N NH 2 N N N H N N HO 5' CH 2 ribose 4' H H 3' OH O H 2' H OH 1' 2 O 3 P O CH 2 H N H OH O H N OH H adenine adenosine adenosine monophosphate (AMP)
Nucleic acids
have a backbone of alternating P i 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.
nucleic acid N NH 2 adenine N 5' end O O P O N N O O 5' CH 2 4' H H O 3' O H 2' H OH 1' ribose P O 5' CH 2 O O H O H O 3' P O NH 2 cytosine N N H OH H ribose O (etc) 3' end O
H cytosine (C) H N N guanine (G) N N H O N N H N H H O NH
G C 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 tRNA Gln . 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 3 rd base. Similar codons tend to code for similar amino acids. Thus effects of mutation are minimized.
1 st base U U
UUU Phe UUC Phe UUA Leu
C A
UUG Leu CUU Leu CUC Leu CUA Leu CUG Leu AUU Ile AUC Ile AUA Ile
G
AUG Met* GUU Val GUC Val GUA Val GUG Val *Met and initiation.
Genetic Code 2 nd base C A
UCU Ser UCC Ser UCA Ser UAU Tyr UAC Tyr UAA Stop 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 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
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
3 rd base 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 A C C
: : : : :
U A U G G G C U U 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 non standard interactions that stabilize tertiary RNA structures.
tRNAs
have an
L-shaped
tertiary structure.
anticodon loop Extending from the
acceptor stem
, the
3' end
of each tRNA has the sequence
CCA
.
tRNA 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
#22 G #46 (m 7 G) #13 C #22 G #46 (m 7 G) #13 C Tertiary base pairs in tRNA Phe Tertiary base pairs in tRNA Phe 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
tRNA Phe with Chime (PDB file 1TRA) .
anticodon tRNA Phe acceptor stem
O R H C C NH 3 + Amino acid O O O O R H C NH 2 C O Aminoacyl
-
AMP O P O O P O ATP O P O O CH 2 H H OH O O O P O O Adenine H OH H CH 2 H H OH O PP i Adenine H OH H
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 a -carboxyl attacks the P atom of the initial phosphate of ATP.
In
step 2
, the 2' or 3' OH of the terminal adenosine of tRNA attacks the amino acid carbonyl C atom.
O O R H C C O NH 2 Aminoacyl
-
AMP P O O CH 2 H H OH O tRNA AMP Adenine H OH H O tRNA O P O O CH 2 H O H O 3’ 2’ OH H C O HC NH 3 + R H Adenine (terminal 3 ’ nucleotide of appropriate tRNA) Aminoacyl -tRNA
Aminoacyl-tRNA Synthetase
- summary: 1.
amino acid + ATP
aminoacyl -AMP + PP i
2.
aminoacyl -AMP + tRNA
aminoacyl -tRNA + AMP
The 2-step reaction is
spontaneous
overall, because the concentration of
PP i
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
coding for that amino acid.
&
tRNAs Identity elements
: tRNA domains recognized by an aaRS.
Most identity elements are in the
acceptor stem
&
anticodon loop
. Aminoacyl-tRNA Synthetases arose
early in evolution
. Early aaRSs probably recognized tRNAs only by their acceptor stems. anticodon loop tRNA acceptor stem
There are 2 families of Aminoacyl-tRNA Synthetases:
Class I & Class II
. O tRNA O P O O (terminal 3 ’ nucleotide of appropriate tRNA) CH 2 O Adenine H H H O 3’ 2’ OH H C O HC NH 3 + R 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
transferred to tRNA. that are misacylated or mis 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 tRNA Ile , to form Val-tRNA Ile , 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
tRNA. Examples: after being linked to a In prokaryotes the initiator
tRNA fMet
is first charged with
methionine
. Methionyl-tRNA formyltransferase then catalyzes formylation of the methionine, using
tetrahydrofolate
as formyl donor, to yield
formylmethionyl-tRNA fMet
. In some prokaryotes, a
non-discriminating aaRS loads aspartate
onto
tRNA Asn
. The aspartate moiety is then
converted
by an amido transferase
to asparagine
, yielding Asn-tRNA Asn .
Glu-tRNA Gln Gln-tRNA Gln
is similarly formed and converted to in such organisms.
Some proteins contain the unusual amino acid
selenocysteine
(
Sec
), with selenium substituting for the S atom in cysteine. H H H 3 N + C COO H 3 N + C COO CH 2 CH 2 SH SeH cysteine selenocysteine 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.
tRNA Sec
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-tRNA Sec
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
aaRS Tyr cytokine
that stimulates
angiogenesis
. yields a A
truncated
form of the human
aaRS Trp
angiogenesis. inhibits Regulation of
apoptosis
by the human
aaRS Gln
dependent on the concentration of its substrate glutamine.
is Several mammalian Aminoacyl-tRNA Synthetases associate with other proteins to form large
macromolecular complexes
being investigated.
whose roles are actively
Ribosome Composition
(S = sedimentation coefficient) Ribosome Source E. coli Rat cytoplasm Whole Ribosome 70S 80S Small Subunit 30S 16S RNA 21 proteins 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 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 mRNA location small subunit 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.
Small ribosomal subunit
of a thermophilic bacterium:
rRNA
in monochrome;
proteins
in varied colors.
30S ribosomal subunit PDB 1FJF spacefill display ribbons 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
.
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. PDB 1FFK Large Ribosome Subunit "Crown" view with RNAs blue, in spacefill; proteins red, as backbone.
The
active site
domain for peptide bond formation is essentially devoid of protein. The
peptidyl transferase
function is attributed to
23S rRNA
, making this RNA a "
ribozyme
." PDB 1FFK Large Ribosome Subunit "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. Nascent polypeptides emerge through a
tunnel
in the large subunit. The tunnel lumen is lined with rRNA helices and some ribosomal proteins.
PDB 1FFK 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
addition to its structural & catalytic roles. in
tRNAs
also undergo substantial
conformational changes
within ribosomal binding sites during protein synthesis.
Explore
the large ribosomal subunit.