Download The genetic code and tRNA Biochemistry 302 February 15, 2006

The genetic code and tRNA
Biochemistry 302
February 15, 2006
Major advances in defining mechanism
of protein biosynthesis
•
Identification of ribosomes
(Paul Zamecnik, 1950s)
– Injected radioactive amino acids
into rats, fractionated liver
homogenates at various time
points, then analyzed subcellular
distribution of labeled proteins.
– At early time points, “hot”
proteins only in “small” RNP
particles.
•
Ribosomes attached to outer face of ER
Identification of tRNAs
– M. Hoagland & Zamecnik
– Amino acids become “activated”
i.e. attached to heat-stable soluble
RNA species if incubated with
cytosolic extract.
– R. Holley → tRNA
•
mRNAguided
protein
synthesis
Crick’s adaptor hypothesis
Predicted triplet code 43 = 64 possible combinations
Overall process of mRNA-guided
protein biosynthesis: translation
H2N
3′
5′
COOH
5′
anticodon
A200-250
7-meG
Fig. 4-22
activated amino acid
or aminoacyl tRNA
Deciphering the triplet genetic code….
Francis Crick and Sidney
Brenner, 1961
• Used growth conditions
which generated mutations in
T4 bacteriophage DNA.
• Deletion or insertion of 1 or 2
nt led to nonsense proteins.
• Deletion or insertion of 3 nt
led to insertion or deletion of
one amino acid residue.
• Code must be in triplets and
read in units of three.
• Eliminated overlapping and
punctuated code hypotheses.
• First codon read establishes
the reading frame.
Fig. 27-1
H.G. Khorana experiments confirm the
code is triplet and unpunctuated
Fig. 27-2
• Developed methods for 1) synthesizing polyribonucleotides
and 2) carrying out cell-free ribosome-mediated translation.
• (AAG)n template gave rise to three different homopolymers.
• Confirmed the importance of the reading frame and revealed
that AAG, AGA, and GAA must be codons for K, R, and E.
~1961-1964 Genetic code is broken
• Heinrich Matthaei, Philip
Leder, and Marshall Nirenberg
•1: Used synthetic RNA
triplets that would bind
ribosomes and specify the
binding of only certain
aminoacylated-tRNAs
• 2: Developed cell-free
translation system using
random RNA polymers
prepared with polynucleotide
phosphorylase
(NMP)n + NDP ⇌ (NMP)n+1 + Pi
• 1+2: Codon assignment
22nd pyrrolysine
21st selenocysteine
Fig. 27-3
Genetic code is not quite universal
(alternate use generally confined to mitochondrial
transcripts in certain organisms)
*
Table 27-1
Important features of the genetic code
• Triplet and non-overlapping
• Redundancy of the code
• Most amino acids are encoded by
several codons, others by only one
(e.g. Met, Trp).
• Generally, degeneracy is found at the
third position.
• Special codons for starting and
stopping protein synthesis
• Start: AUGMet (uncommon: UUGLeu,
AUUIle, GUGVal)
• Stop: UAA, UGA, UAG (rare alternate
use: UGASec, UGATrp, UAGPyl)
• Open reading frame
• Biochemical basis for complexity
of the code
• Base-pairing between tRNA anticodon
and mRNA codon
• Optimize speed and accuracy of
protein synthesis
Note antiparallel
alignment of tRNA
and mRNA
Wobble hypothesis: attempt to explain
redundancy of the genetic code
• Rationale for why single a
tRNA can recognize several
different codons.
• Multiple recognition involves
the 3′ residue of the codon and
the 5′ residue of the anticodon.
• In 1966, Crick proposed that
the 5′ base of the anticodon
could wobble during translation.
− Movement permits nonWatson-Crick H-bonding.
− Model explains frequent
degeneracy observed in the
3′ site of some codons.
e.g. AlatRNAAla
can base
pair with
codons
GCC and
GCU.
Crick’s wobble rules (base-pairing
possibilities in wobble pairs)
• First 2 base pairs in codon-anticodon
interaction form strong WC H-bonds
and, usually specify the amino acid.
• First base (5′) of anticodon
determines # of codons read:
• C, A → only one codon read by tRNA,
binding is specific.
• U, G → two codons read by tRNA,
binding a little less specific.
• I → three codons read by tRNA
(tRNAArg), “weak” base-pairing
• Codons that specify the same amino
acid but differ in either of the first two
bases require different tRNAs (e.g.
Arg, Ser, Leu)
• Rules require a minimum of 32 tRNAs
to translate all 61 amino acid codons
(∼70 in E. coli).
Table 27-2 Wobble bps
Wobble hypothesis does not explain
all complexities inherent in decoding
•
•
•
tRNAs whose anticodons
(e.g. CUA, UCA) decode stop
codons: UAG (pyrrolysine),
UGA (selenocysteine)
Selenocysteine found in
formate dehydrogenase
(bacteria), glutathione
peroxidase & thioredoxin
reductase (mammals).
Alternate use of stop codon
dictated by…
– Specific 3′ mRNA hairpin
element (SECIS) and binding
protein (SBP2)
– Specialized elongation factor
known as (eEFSec or SelB)
– Sec-tRNASec (actually special
type of serine tRNA that gets
converted to Sec)
Pyrrolysine
L-lysine in amide linkage
(4R,5R)-4-substituted
pyrroline-5-carboxylate
Overview of tRNA structural elements
and interaction partners
•
•
73 to 93 nucleotides long
(eukaryotes) and arranged in
a canonical 2-D cloverleaf
(tRNAAla R. Holley, 1965)
Intrastrand H-bonding
specifies structure
– Four “arms” and a “stub”
– pG on 5′ end, CCA acceptor
stem on 3′ end (all tRNAs)
•
Interaction partners
–
–
–
–
•
Processing enzymes
Aminoacyl-tRNA synthetases
EF-Tu (elongation factor)
Ribosome
Where does specificity come
from in 73 to 93 nucleotides?
76 residue yeast tRNAAla 1st nucleic
acid to be completely sequenced.
Note the number of modified bases.
General secondary and tertiary
(twisted L) structure of tRNAs
3-D Structure of
yeast tRNAPhe
deduced from xray diffraction
Invariant residues are
boxed and shaded in pink.
The D- and TψC-arms
are important for tertiary
folding & geometric
separation of acceptor
stem & anticodon loop.
The TψC arm interacts
with the large
ribosomal subunit.
Many modified bases are commonly
found in tRNA
no double
bond
N and C in
altered
positions
ψ
Fig. 27-7
•
•
•
Provide increased stability by facilitating proper 3-D folding.
Modify the specificity of anticodon-codon interactions
(inosine).
Serve as recognition elements for amino acyl-tRNA
synthetases.
Interactions mediating conserved 3-D
tRNA folding (hand drill or twisted L)
•
•
The D loop and TψC
loops fold inward to
provide maximum Hbonding and base
stacking.
Fig. 4-20
Yeast tRNAPhe 76 bases
•
Single-stranded but
extensive base pairing
(self-complementary) and
base stacking
Two A-form helices at right
angles to each other
Non Watson:Crick
interactions:
– Unusual base pairs
– Base triples
– Base backbone contacts
Conservation of 3-D structure is to
ensure correct fit in the ribosome.
Aminoacylation of tRNA adaptor, the
first step in protein synthesis
aaRS
Mg++
Amino acid + ATP
Amino Acid~AMP + PPi
aaRS
+tRNA
Amino Acid~AMP
aaRS: aminoacyl-tRNA synthetase
(21 synthetases in E. coli each
recognizing one AA and one or more
tRNAs. Lys has two aaRSs. aaRSs
also have hydrolytic “proofreading”
ability.)
AA-tRNA + AMP
Chemistry of aminoacylation….
Enzyme
bound
Class II aaRS: AA is
transferred to the 3′
OH of invariant
adenosine residue on
acceptor arm.
Class II aaRS
Class I aaRS: AA is
transferred to the 2′
OH of invariant
adenosine then to
the 3′ OH via
transesterification.
Class I aaRS
Fig. 27-10
How an aminoacyl-tRNA synthetase
identifies the right tRNA….no simple rule,
depends on each enzyme, “second genetic code”
Recognition
elements exist at
many different
locations but tend to
cluster in the
anticodon loop and
acceptor stem.
Fig. 27-11
Structural features of aa-tRNA synthetases
Acceptor
stem, ATP,
& AA in
close
proximity
•
•
•
•
Class I: glutaminyl-tRNA
synthetase (monomers)
Class II: aspartyl-tRNA
synthetase (dimers/tetramers)
(Met, Ile, Val, Leu, Tyr, Trp, Glu,
Arg, Gln, Cys, Lys)
(Ser, Pro, His, Thr, Gly, Asp, Asn,
Lys, Ala, Phe)
Different protein folds
•
Different consensus sequences for •
ATP binding
Different mode of tRNA recognition •
Different stereochemistry
•
Subgroups: tRNA binding domains
Examples of class switching (e.g.
TyrRS binds tRNA in class II mode)
Induced fit facilitates catalysis
Hydrolytic proofreading