Chapter 20
Protein Synthesis and Sorting
+++++Rough Draft of Term Paper is due Friday the 13th of April+++++
I. Translation = protein synthesis
A. Required Components
1. Ribosomes
a. two types
(1) 80S (eukaryotes)
- 40S small subunit
- 60S large subunit
(2) 70S (bacteria, mitochondria, chloroplasts)
- 30S small subunit
- 50S large subunit
b. composed rRNA and many proteins
(1) many structural proteins
(2) some enzymatic proteins
(3) see Fig 20-1 & Table 20-1
c. three tRNA binding sites
(1) P site (peptidyl)
(2) A site (aminoacyl)
(3) E site (exit)
(4) Fig. 20-2
d. one mRNA binding site
e. mRNA orientation site
-see below
f. X-ray structure (low resolution)
now available
(1) green = aminoacyl tRNA
(2) blue = peptidyl tRNA
(3) red = exit tRNA
g. higher resolution (2.4 A) structure of the large
subunit now available at
Science 289:905-920 (2001)
2. tRNA's
a. crystal structure of yeast tRNA
(1) wireframe
(2) spacefilling
a. tRNAs accomplish the "translation" of
nucleic acid language (the 4 bases) into
protein language (20 amino acids)
b. each tRNA has an anticodon which is
complementary to a specific codon
of mRNA
(1) see Fig 20-3
c. each "charged" tRNA carries specific
covalently attached amino acid
appropriate to the codon
(1) amino acids are attached by
specific enzymes
(a) aminoacyl-tRNA-transferases
(2) amino acids are esterified to the 3' -OH
of the A of the CCA terminus
d. 61 different codons for amino acids
e. there are not 61 different tRNA's
(1) when anticodon associates with codon,
3rd position is less definite
-3rd position = the 5' end of anticodon
-recall: mRNA is read 5' to 3'
-recall: anticodon is complementary
and antiparallel to codon
-therefore anticodon is read 3' to 5'
-3rd position can "wobble"
(2) usually, first two bases most important
in determining the amino acid
see the table of codons
(3) tRNA's (anticodons) often have inosine
as the base in 3rd position
(a) inosine is "wobbliest" base
(b) inosine can base pair with U, C or A
(c) inosine is like an adenosine with a -OH
substituted for the -NH2
(c) see Fig. 20-4
f. due to wobble, fewer than 61 tRNA's are
needed
-cell benefits by needing one or two tRNA
genes for each amino acid instead of
one for each codon
-what amino acid is associated with this tRNA?
-table of codons
-what other anticodon is required for this amino acid?
-what about 3'-GCC?
3. aminoacyl-tRNA synthetases
a. see Fig. 20-5
b. enzymes link amino acids to
cognate tRNAs
c. see crystal structure of asp-tRNA synthetase
c. linked by ester bond to 3'-OH of ribose
d. critically important enzymes in cell
-must be very accurate at attaching the
right amino acid to the right tRNA
-What phenotype is expressed when one tRNA
synthetase doesn't work?
-What phenotype is expressed when one tRNA
synthetase works with 50% efficiency?
4. mRNA = messanger RNA
a. see chapter on transcription
b. mRNA has the coding information needed to
synthesize the new protein
5. protein factors
a. various protein factors required for translation
(1) initiation factors
(a) IF1 (initiation factor 1)
(b) IF2 (initiation factor 2)
(c) IF3 (initiation factor 3)
(2) elongation factors
(a) EFtu (elongation factor-Tu)
(b) EF-G (elongation factor-G)
(3) termination factors
(a) release factors
B. Steps in Translation
1. initiation
a. see Fig 20-8
b. start with large and small subunits,
tRNAs, mRNAs and initiation factors
free in solution
c. the 70S initiation complex forms
(1) IF-1, IF-2, IF-3 & GTP bind to 30S
subunit
(2) initiator t-RNA and mRNA bind to 30S
ribosome
(3) IF-1 and IF-3 fall off
(4) 50S subunit binds to complex
with the initiator t-RNA on the P site
(5) GTP is hydrolyzed to GDP + Pi and
EF-2 falls off ribosome
(6) 70S ribosome is complete and ready to
elongate a polypeptide chain
d. reading frame determination
(1) this is part of the initiation process
(2) every mRNA has a 5'-AGGA-3' sequence just
upstream from the reading frame
(a) reading frame starts with 5'-AUG-3'
(3) AGGA binds to a complementary
sequence (3'-UCCU-5') on the 3' end of
the 16S rRNA of the 30S subunit
(4) initiator tRNA carrying N-formylmethionine
(f-Met) recognizes the codon AUG and
binds in a complementary fashion
at the part of the P-site on the 30S
subunit
(a) f-Met-tRNA is only tRNA which can
bind to the part of the P-site on 30S
subunit to initiate protein synthesis
(b) NOTE: the formyl group blocks
the reactivity of what would otherwise
be a free amino group
(c) f-methionine can't be used except as
the "N"-terminal amino acid
2. chain elongation
a. see Fig. 20-10
b. tRNA with anticodon specific for codon under
A-site binds at A-site
(1) EFtu with bound GTP binds charged tRNA
(2) EFtu-tRNA complex binds at A site
(3) GTP hydrolyzed to GDP + Pi
(4) EFtu, GDP and Pi leave
(5) tRNA stays on the A site
c. now two tRNAs lie side by side
d. f-Met close to other amino acid connected
to a tRNA at A-site
e. f-Met transferred to the amino acid on
the tRNA at the A-site
(1) ester bond between f-Met and tRNA is hydrolyzed
(2) now free carboxyl of f-Met forms peptide bond
with free amino group of the amino acid
esterified to the tRNA occupying the A site
(3) catalyzed by peptidyltransferase
(4) a ribozyme = peptidyl transferase that is
part of the rRNA of the large subunit of the ribosome
catalyzes the formation of a peptide bond
between the formylated-methionine (or growing peptide chain)
associated with the P site and the
amino acid associated with the A site
f. now tRNA occupying the P-site has no
amino acid
g. EF-G-GTP binds to EXIT site
h. EF-G-GTP pulls mRNA, tRNAs
on the A site and P site
over 1 codon (3 bases)
-all codon-anticodon associations remain
intact during translocation
-GTP gets hydrolyzed to GDP + Pi
-EF-G, GDP, Pi all fall off
i. tRNA over the EXIT site falls off
l. now the A site is empty
m. next appropriate tRNA binds to A site
-EFtu involved again
n. dipeptide is transfered to the -NH2 group
of the amino acid covalently attached to the
tRNA on the A site
o. back to step "i"
p. these steps are repeated until a "STOP" codon
3. chain termination
a. see Fig. 20-11
b. tRNA covalently attached to the finished
protein gets to the P site
b. "STOP" sequence appears under the A-site
(1) UAG, UAA or UGA
(2) no corresponding tRNA
c. release factors recognize stop sequence and hydrolyze
finished protein from tRNA
d. tRNA w/o peptide falls off ribosome
e. whole translation assembly disassembles
4. Quick Review of Translation
a. initiation
-required components
-completed assembly
b. elongation
-second tRNA binds
-formation of the dipeptide
-move to the P site
-third tRNA binds
-formation of the tripeptide
c. termination
-arrive at the "STOP" codon
-release factor removes the completed protein
-translation complex disassembles
II. Protein Targeting and Sorting
A. How does each protein get to its correct intracellular
destination?
B. group the various destinations
1. proteins needed in cytoplasm
2. proteins needed in organelles
a. mitochondria
b. chloroplasts
c. peroxisomes
d. nucleus
3. proteins needed in endomembrane system
a. ER
b. nuclear envelope
c. Golgi
d. lysosomes
e. secretion vesicles
f. plasma membrane
C. sorting process
1. start with nuclear genes
2. mRNA made in nucleus
3. mRNA goes to cytoplasm through
nuclear pore
4. all protein synthesis begins on
cytoplasmic ribosomes in cytoplasm
a. cytoplasmic ribosomes not bound to
any membrane
5. once translation starts, divergence begins
a. ribosomes synthesizing proteins destined
to ER membrane or lumen of ER become
attached to RER shortly after protein
synthesis starts
(1) called "cotranslational import"
(2) where will these proteins end up?
-think about membrane flow theory
-how do proteins get into lysosomes?
-how do proteins get to PM?
-how do proteins get secreted?
b. cytoplasmic ribosomes which remain
free in cytoplasm
(1) are synthesizing proteins destined for
(a) cytoplasm
(b) nucleus
(c) chloroplasts
(d) mitochondria
(e) peroxisomes
(2) proteins are first released free into cytoplasm
after translation
(3) proteins then taken up by chloroplasts, nucleus,
mitochondria or peroxisome if appropriate
(a) must have the appropriate
transit sequence of amino acids
(b) called "posttranslational import"
(4) see details and cartoons below
D. cotranslational import of proteins
1. "signal mechanism" (Blobel and Sabatini)
a. applies to all proteins which are synthesized
on ribosomes attached to ER
b. Blobel and Sabatini developed a
hypothesis (model) which
described how proteins were inserted
into ER membrane and lumen
(1) originally called the "signal hypothesis"
(2) now so well established that it is called
the signal mechanism
2. N-terminus is first part of protein
synthesized
3. "signal" = first 15-30 amino acids of N-terminus.
a. first some positively charged
amino acid R groups at N-terminus
b. then about 12 amino acids with
hydrophobic R groups
5. only proteins with an appropriate
"signal" will:
bind to ER membrane and be
put into ER lumen or be put
into ER membrane with one orientation or
into ER membrane with another orientation
a. now, with recombinant DNA techniques,
can add a signal sequences to any
desired protein, thus sending the protein
into ER lumen for subsequent excretion
outside of the cell or targeting the protein
for important into any organelle!
7. binding of signal to ER membrane
a. see Fig 20-16 a, b
b. signal-recognition particle (SRP)
(1) SRP binds to signal as soon as it is
synthesized
-SRP binding blocks further
translation until membrane
"docking" occurs
c. SRP+ribosome binds to SRP receptor on
ER membrane ("docking")
d. GTP binds to SRP receptor and
signal moves into pore protein
e. GTP --> GDP + Pi and SRP falls off
(1) note that energy (GTP) has been
used to "defy entropy" and to make
the system more organized
(2) without bound SRP to stop translation,
translation resumes
(3) signal remains bound in pore protein
(4) "loop" of newly synthesized protein forms
f. polypeptide elongates as ribosome continues
to translate mRNA into protein
(1) same elongation steps as detailed above
g. as the C-terminus of the protein
passes through the pore,
a peptidase cleaves off
finished protein which is now free
in the lumen of ER
8. synthesizing integral proteins with
stop transfer sequence
a. see Figure 20-17
b. start with normal ER signal sequence
c. uses SRP to facilitate binding
to SRP receptor
d. elongation proceeds as above
e. toward the C-terminal end of protein
is a stop transfer sequence
-transfer through pore stops
-translation of rest of protein continues
-C-terminus remains on cytoplasmic side
as a cytoplasmic domain
-protease removes the signal
-integral protein "floats" free in membrane
9. synthesizing integral proteins without
stop transfer sequence
a. special "internal signal sequence" =
"start transfer sequence"
b. SRP binds to "start transfer sequence" and
delivers to the membrane
c. N-terminus stays on the cytoplasmic side
as a cytoplasmic domain
d. "start transfer sequence" enters pore,
forms a loop, and binds to pore wall
d. loop grows into the pore
e. no "stop transfer sequence", so rest of
protein passes through membrane
f. protein floats free in the membrane
with C-terminus exposed to lumen of
ER and N-terminus exposed to cytoplasm
E. posttranslational import of proteins
1. see Fig. 20-19
2. N-terminus of protein has a
"transit peptide"
3. transit peptide directs protein to
specific protein transporter
in membrane of target organelle
a. no SRP is needed to allow the transit
peptide to bind to the protein transporter
b. BUT "chaperone" proteins
are needed to keep the protein in a
"unwound" condition while in the cytoplasm
4. when transit peptide passes through the
transporter, it is
cleaved off before rest of the protein follows
-remaining protein passes through the
transporter
-cytoplasmic chaperone proteins stripped
off as protein passes through transporter
-mitochondrial chaperones added inside matrix
5. cleavage of first "transit peptide" may reveal
another transit peptide which
sends the protein through next membrane
a. eg: proteins for thylakoid membrane
(1) first transit peptide brings protein
through the two membranes of
the envelope
(2) second transit peptide and second
protein transporter inserts the protein
into the thylakoid membrane
6. final protein functions without transit peptide or
chaperones
F. Do Formative Assessments on Translation
.
Components of Ribosomes
| S Value | Subunit | Subunit S Value | Proteins | rRNA |
| Prokaryotic | 70S | Large | 50S | 34 | 23S & 5S |
| | Small | 30S | 21 | 16S |
| Eukaryotic | 80S | Large | 60S | 45 | 28S, 5.8S & 5S |
| | Small | 40S | 33 | 18S |
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