Transcript Genomes 3/e - Illinois Institute of Technology

Chapter 13:
Synthesis and Processing
of Proteome
Copyright © Garland Science 2007
Transcriptome expression
• Synthesis of the proteome (tRNAs
decode, polymerization in ribosome)
• Processing of the proteome (folding,
cutting, chemical modifications)
• Degradation of the proteome.
13-1. tRNA &
protein synthesis.
tRNAs are adaptor
molecules between
mRNA and polypeptide
Both physical
(amino-acylation) &
informational (codonanticodon recognition)
Isoaccepting tRNAs
specific for same AA (3050 tRNAs vs. 20 AA)
Figure 13.1 Genomes 3 (© Garland Science 2007)
13-1. tRNA &
protein synthesis.
tRNAs 74-90 nt in length;
cloverleaf structure.
Acceptor arm attaches
amino acid; anticodon
arm attaches mRNA; 3
other arms are conserved.
Some positions are
completely invariant;
important for tertiary
structure stability.
Figure 13.2-3 Genomes 3 (© Garland Science 2007)
13-1. tRNA &
protein synthesis.
Aminoacyl-tRNA
synthetase catalyzes
transfer of amino acid
to 2’ or 3’ –OH of tRNA
20 synthetases in Class
I (2’-OH) & Class II (3’OH) highly specific to
amino acids.
Figure 13.4 Genomes 3 (© Garland Science 2007)
13-1. tRNA &
protein synthesis.
Attachment of tRNA to
mRNA is based on
codon-anticodon
interactions by basepairing.
Wobble effect due to
the curved shape of
anti-codon may allow
non-standard base
pairing (e.g. G-U & 3’UAI-5’ in bacteria; 16
of 48 human tRNAs
read 2 codons).
Figure 13.6 Genomes 3 (© Garland Science 2007)
13-2. Ribosome in
protein synthesis.
E. coli has 20,000
ribosomes in
cytoplasm; human has
even more; complex of
rRNAs + proteins
Functions include to
coordinate protein
synthesis by placing
mRNA, tRNA, proteins
in correct positions;
catalyze some
translation reactions.
Figure 13.12 Genomes 3 (© Garland Science 2007)
Sedimentation
coefficient by
ultracentrifugation
Figure 13.10 Genomes 3 (© Garland Science 2007)
13-2. Ribosome in protein synthesis.
In E. coli, ribosome is assembled on mRNA at
initiation codon w/translation initiation factor IF-3
(prevents premature dissociation); 3’ of 16S rRNA
attached to ribosome binding site (Shine-Dalgarno
sequence).
Figure 13.14 Genomes 3 (© Garland Science 2007)
13-2. (Cont.)
Translation
initiation in
bacteria.
Initiation codon AUG
(Methionine); initiator
tRNA is modified by
attaching –COH to Met N
terminal (fM); IF-2 & GTP
are used by large subunit
to bind; internal AUG is
recognized by a different
tRNAMet w/unmodified Met.
Figure 13.15 Genomes 3 (© Garland Science 2007)
13-2. (Cont.)
Translation initiation
in eukaryote.
Most mRNAs don’t contain
ribosome binding sites
(unlike bacteria);
preinitiation complex (40S)
is first assembled prior to
binding; eIF-2 binds GTP &
unmodified tRNAMet; cap
binding complex acts as a
bridge in between; binding
also affected by poly(A) via
PADP, a poly(A) binding
protein.
Figure 13.16a Genomes 3 (© Garland Science 2007)
13-2. (Cont.)
Translation
initiation in
eukaryote.
Preinitiation complex
scans along mRNA until
it reaches the initiation
codon (a few tens or
hundreds nt
downstream & located
within Kozak consensus
sequence);
large subunits then
attach.
Figure 13.16b Genomes 3 (© Garland Science 2007)
13-2. (Cont.)
Regulation of
translation
initiation.
Global regulation (e.g.
under stressful
conditions) by eIF-2
phosphorylation
prevents GTP binding,
therefore represses
translation; transcript
specific regulation by
feedback inhibition or
feedback activation
mechanisms (left)
Figure 13.17a Genomes 3 (© Garland Science 2007)
13-2. (Cont.)
Elongation
Large subunit has 2 sites P
site (peptidyl site)
w/tRNAMet; A site
(aminoacyl site) w/tRNA for
the next codon.
Elongation factor EF-1
ensures accuracy of new
tRNAs; peptidyl transferase
forms new peptide bond;
EF-2 translocates the new
tRNA & opens up A site.
Figure 13.18 Genomes 3 (© Garland Science 2007)
13-2. (Cont.)
Frame-shifting during elongation
Ribosome pauses spontaneously & moves back for
1 nt & continues translation: changes the reading
frame; 3 types of frame-shifting: programmed
frame-shifting enables translation of multiple
proteins from the same mRNA
Figure 13.21a Genomes 3 (© Garland Science 2007)
13-2. (Cont.)
Frame-shifting during elongation
Translation slippage: enables a single ribosome to
translate an mRNA that contains copies of 2 or
more genes. Similarly, translational bypass.
Figure 13.21b-c Genomes 3 (© Garland Science 2007)
13-2. (Cont.)
Termination
At the termination
codon, A site is
occupied by a protein
release factor;
ribosome
disassociates by
ribosome release
factor (RRF).
Figure 13.22 Genomes 3 (© Garland Science 2007)
13-3. Post-translational processing
Four major types of processing:
Figure 13.24 Genomes 3 (© Garland Science 2007)
13-3. (Cont.)
Protein folding
Four levels of protein
structure; need correct
tertiary structure to be
activated; a dynamic
process; for large
proteins, renaturation is
not always spontaneous
due to (1) tendency to
form insoluable
aggregates; (2) more
stable alternative folding
pathways.
Figure 13.25-26 Genomes 3 (© Garland Science 2007)
13-3. (Cont.)
Protein folding
Protein folding is
assisted by molecular
chaperons (to hold
proteins in an open
conformation for folding)
& chaperonins (a protein
complex to promote
folding through a cavity
& proof-read incorrectly
folded proteins into
correct folding).
Figure 13.27-28 Genomes 3 (© Garland Science 2007)
13-3. (Cont.)
Proteolytic
cleavage
Protein cutting is
either end-processing
(to cut off N or C
terminals to make
functional proteins)
or poly-protein
processing (to cut
into small pieces of
functional proteins).
Figure 13.29 Genomes 3 (© Garland Science 2007)
13-3. (Cont.)
Proteolytic
cleavage
An example of endprocessing is pre-proinsulin. Step 1. Cut
off 24 amino acids
from N terminal to
give pro--insulin;
step 2. Cut internal B
chain to give insulin.
Figure 13.31 Genomes 3 (© Garland Science 2007)
13-3. (Cont.)
Proteolytic
cleavage
An example of polyprotein processing
used as a way to
reduce size of
genomes w/ a single
gene & 1 promoter &
1 terminator; can be
spliced in various
ways in different
cells.
Figure 13.32 Genomes 3 (© Garland Science 2007)
13-3. (Cont.) Chemical modification
Table 13.6 Genomes 3 (© Garland Science 2007)
13-3.
(Cont.)
Chemical
modification
More complex
modification is
glycosylation
used to add
large
carbohydrate
side chains to
Serine (Olinked) or
Asparagine (Nlinked).
Figure 13.34a Genomes 3 (© Garland Science 2007)
13-3. (Cont.)
Intein splicing
A protein version of
RNA splicing (vs.
extein); first discovered
in yeast in 1990; also
found in bacteria &
archaea; most ~150 aa
& self-catalyzed; intein
homing (convert a
intein- gene into a
intein+ gene; used as a
mechanism to
propagate).
Figure 13.35 Genomes 3 (© Garland Science 2007)
13-4. Protein
degradation
Proteolysis by
proteases is dependant
on degradationsusceptibility signals;
in eukaryotes,
proteasome unfolds
proteins & cuts into 410 aa; released to
cytoplasm & further
broken down to
individual amino acids.
Figure 13.37 Genomes 3 (© Garland Science 2007)
Chapter 13 Summary
End result of genome expression is proteome (a
collection of proteins in a cell); tRNA 3’ end is
attached to amino acid by aminoacylation; 5’ end
is attached to mRNA by condon-anticodon
interactions; wobble effect allows single tRNA read
more than 1 codons.
Bacterial ribosome has internal binding site for
mRNA; eukaryote doesn’t; initiation is controlled
by global or transcript-specific mechanisms;
unusual elongation includes programmed reading
frame-shifting and translation bypassing; proteins
are processed by proteolytic cleavage or chemical
modifications & degraded by proteasome.