| M W F; 10:10 - 11:00 pm |
|
Douglas W. Smith |
| York 2722 |
|
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
Readings: Brown, 10: 232-235; 10: 240-244
Outline:
A. Basics of Translation or Protein Biosynthesis:
Initiation: complex of mRNA, charged initiation tRNA, and small and large ribosomal subunits is formed
Elongation: complete ribosome moves along mRNA, 5' to 3', synthesizing polypeptide chain one amino acid per charged tRNA, N-terminal end to C-terminal end
Termination: stop codons (UAG, UAA, UGA) signal release of polypeptide chain, mRNA, final tRNA, separation of complete ribosome into small and large subunits
Evidence that proteins are
synthesized on ribosomes via pulse-labeling experiments:
Pulselabel with radioactive amino acids: counts appear first on
polysomes in prokaryotes, and then subsequently on released free
proteins ...
Post-Translational Modifications:
Proteolytic Cleavage
(polyProtein cleavage in viruses; excision of inteins)
Chemical Modifications (glycosylation; phosphorylation; di-Sulfide bonds)
Protein Folding ... Chaperone assistance
B. Components: mRNA, Ribosome small subunit, Ribosome
large subunit, tRNA, Factors
Factors: proteins; small molecules, e.g. ATP and GTP
C. tRNA:
1. Adaptor hypothesis of
Crick: ... [Brown,
Fig 10.1]
adapt amino acid to codon ... amino acids can not steriochemically
fit directly to codons
Most organisms have close to
One tRNA per Codon
For example, E. coli encodes some 86 tRNA species and the yeast
(S. cereviseae) genome contains some 275 tRNA genes (some are
from the mitochondrial genome)
2. tRNA Structure:
1° structure: small, 75 nucleotides, many modified bases: Di-hydroU; Pseudo-U: Y; modified G's; Thymine ... [Brown, Table 9.6, Fig 10.2]
2° structure: cloverleaf
of 4 major stem-loops, 1 minor stem-loop
names of arms: D-arm (dihydro arm), AminoAcyl or Acceptor arm,
T(pseudoU)C arm, Extra arm (Type 1: short; Type 2: long), Anticodon
arm ... [Brown, Fig 10.3]
3° structure: L-shape, with formation of additional H-bond interactions, some of which are nonstandard W-C base interactions ... [Brown, Fig 10.3]
3. tRNA Function:
1) bring amino acids to the ribosomes for protein synthesis via
covalent linkage of amino acid to the tRNA: aminoacyl-tRNA species
2) anticodon of tRNA H-bonds with codon of mRNA via standard
W-C interactions plus non-standard H-bonds resulting from Wobble
(see below)
Function of each tRNA arm:
... [Brown, Fig 10.3]
1) AminoAcyl or Acceptor arm: covalently bind the amino acid;
interact with aminoacyl tRNA synthetases; provide substrate for
peptidyl transferase in the large subunit ribosome.
2) D-arm (Dihydro arm): important in 3D structure of tRNA; interacts
with large subunit ribosome in P and A sites
3) Anticodon arm: provide anticodon for H-bond interactions with
codons in mRNA; interact with aminoacyl tRNA synthetases; interact
with P and A sites in small ribosome subunit.
4) T(pseudoU)C arm: important in 3D structure of tRNA; interact
with large ribosome subunit, particularly in region of this arm
where the sequence is conserved between tRNA molecules.
5) Extra arm: important in 3D structure of tRNA.
4. Aminoacyl-tRNA Synthetases: enzymes which catalyze covalent linkage
of amino acid to a tRNA ... this charges or activates
the tRNA, yielding an Aminoacyl-tRNA species
... [Brown, Fig 10.4]
a. Two-step Catalytic Reaction:
1) ATP + aa --> aa-AMP + PPi, and PPi --> Pi + Pi
NH3+
| O-
where aa-AMP is: R-C-CO-O-P-O-Ribose-Adenine
H O
2) aa-AMP + tRNA --> aa-tRNA + AMP
b. Two classes of Aminoacyl-tRNA
Synthetases: bind to
opposite "sides" of the tRNA
... [Brown, Table 10.1]
Class I: binds minor groves of acceptor and anticodon stems; ATP binding domain a nucleotide binding domain at N-term end of enzyme; anticodon arm binding domain at C term end; synthetase attaches aa to the 2'-OH of the ribose of the 3 terminal nucleotide (the A of the -CCA) of the tRNA
Class II: binds major groves of acceptor and anticodon stems; anticodon arm bound in major groove by N-term domain; ATP binding domain more C-term; synthetase attaches aa to the 3'-OH of the ribose of the 3 terminal nucleotide (the A of the -CCA) of the tRNA
c. tRNA "Identity"
Elements
Most organisms encode
close to One Synthetase per amino acid
For example, the E. coli genome contains some 22 synthetase
genes and the yeast genome contains some 27 synthetase genes.
Problem: If there are roughly 1 tRNA species per codon but only one Synthetase per amino acid, then a Synthetase for an amino acid which has more than one codon must be able to specifically recognize and bind each of the tRNA species for each of these codons.
Example: Arginyl-tRNA synthetase must be able to bind each of the six Arginine tRNA species ... but no other tRNA species.
How is this accomplished?
Answer (in part ... much is still unknown): tRNA species that
encode a given amino acid contain Identity Elements. These
are nucleotides found in common between these tRNA species, but
which are, at least in part, different in all other tRNA species.
Identity Element Nucleotides:
These are found primarily in two locations:
1) the Anticodon
2) the 5'-terminal nucleotides and the 3'-terminal nucleotides
adjacent to the -CCA residues
Some are also found in the D-loop stem-loop ...
d. Discrimination: correct tRNA, correct aa, correct aa-tRNA formed
The correct amino acid must be joined to the correct tRNA by the Synthetases. No subsequent step in protein biosynthesis checks that this has been done correctly
Synthetases use three Proofreading steps to insure that the correct amino acid has been loaded onto the correct tRNA ... the resulting error rate is < 10-5
1) tRNA proofreading:
cognate tRNA binds rapidly, dissociates slowly, triggers conformational
change in enzyme
incorrect tRNA binds rapidly, dissociates RAPIDLY
2) aa proofreading: wrong aa
1st proofreading: wrong aa is bound and attached to AMP
=> aa-AMP is often hydrolyzed
2nd proofreading: wrong aa is acylated to correct tRNA => aa-tRNA
is hydrolyzed
5. Wobble hypothesis: codon-anticodon recognition ... [Brown,
Fig 10.9]
Degeneracy or Redundancy in the genetic code occurs primarily
in the 3rd position of the codon. To account for this, Crick proposed
the Wobble Hypothesis. This hypothesis states that base pairing
of bases at the 3rd position of the codon and the 1st position
of the anticodon can be nonstandard due to "wobble"
of the nucleotide at the 1st position of the anticodon. Such wobble
is due to the curvature of the anticodon in the loop of the anticodon
arm of the tRNA.
Often one tRNA can recognize more than one codon => "wobble" in pairing at 3rd position of Codon ... or ... wobble in pairing at 1st position of AntiCodon ...
Pairing Rules of Wobble
Hypothesis:
U <-> G: U in
Anti -> A or G; C -> G only; A -> U only; G -> C or
U
I <--> C, U, A
I = Inosine: base which is identical to Guanine but lacking the amino group
Examples of such pairings: ... [Brown, Fig 10.9]
6. Suppressor tRNAs: mutated AntiCodons
Suppressor tRNAs suppress mutations present in an mRNA during
translation by inserting an "incorrect" amino acid into
the growing polypeptide chain.
Types of such suppression:
suppression of nonsense,
missense, frameshift mutations ... see [Brown, Research Brief
10.1]
Example of Suppression: a second mutation (in the anticodon nucleotides of a tRNA gene) which suppresses the effects of a first mutation (in the gene being translated)
1) Nonsense suppression: amber, ochre, opal mutants in the
mRNA
Amber mutation: sense codon mutated to UAG
Ochre mutation: sense codon mutated to UAA
Opal mutation: sense codon mutated to UGA
Each of these mutations yields a Stop Codon in the middle of a gene, resulting in synthesis of a truncated (shortened) polypeptide chain. Such polypeptides are nearly always inactive as enzymes.
A suppressor tRNA recognizes the mutant stop codon, and inserts an amino acid in this position of the polypeptide during translation. The resulting non-truncated polypeptide will be enzymatically active if it can tolerate the amino acid change at this position.
Most suppressor tRNAs are minor tRNAs which recognize codons that are used infrequently, and most suppressor tRNAs only suppress a small fraction of the time (1-6% suppression). Otherwise, they would "suppress" at normal Stop Codons, resulting in an abundance of abnormally long, nonfunctional proteins.
2) Missense suppression: similar to nonsense suppression but suppression of a missense mutation
3) Frameshift suppression: frameshift mutants are not suppressed by suppressor tRNAs. However, they can be suppressed by slippage of the ribosome by one nucleotide. Such is usually done by mutant ribosomes. Hence, the suppression is by a second mutation in a ribosomal protein gene.
D. Ribosomes: ... [Brown, Fig 10.10]
1. Ribosome Structure:
Prokaryotes: 30S small subunit + 50S large subunit -> 70S complete
ribosome
Higher Eukaryotes: 40S small subunit + 60S large subunit ->
80S complete ribosome
rRNA: ... [Brown, Fig 10.11, Fig 10.12]
Prokaryotic 30S ribosome: 16S rRNA species
Prokaryotic 50S ribosome: 5S and 23S rRNA species
Eukaryotic 40S ribosome: 18S rRNA species
Eukaryotic 80S ribosome: 5S; 5.8S, 28S, 5.8S is H-bonded to the
28S species
Proteins:
Prokaryotic 30S ribosome: 21 proteins
Prokaryotic 50S ribosome: 31 proteins
Eukaryotic 40S ribosome: 33 proteins
Eukaryotic 80S ribosome: 50 proteins
3D structure:
complex structure, with protuberances, as seen by microscopy
structure now known to 6 Angstrom atomic resolution for Prookaryotic
30S ribosome ...
Much rRNA stem-loop structure
2. Ribosome assembly:
Discrete sub-particles have
been isolated
"Split" proteins have been isolated by banding ribosomes
in CsCl gradients ...
"Split" proteins are removed by the CsCl treatment
Assembly proceeds in step-wise coordinated fashion as the larger rRNA molecule is transcribed
3. Ribosome Binding Sites:
... [Brown, Fig 10.16]
3 sites for tRNA: A site (entry site), P site (aa transfer),
E site (exit)
mRNA site on 30S ribosome
Peptidyl transferase site on 50S ribosome
5S rRNA site on 50S ribosome
If you have problems or comments, send email to Doug
Smith