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1052 Chapter 27 Protein Metabolism

O O O
H B B B
 
ROC CO O  O O P OO O P OO O P O OOCH2
A B A A A

NH3 O O O O
O Adenine
Amino acid ATP
H H
H H

OH OH MECHANISM FIGURE 2714 Aminoacylation


of tRNA by aminoacyl-tRNA synthetases. Step
1 1 is formation of an aminoacyl adenylate,
PPi
which remains bound to the active site. In the
second step the aminoacyl group is transferred
O
H B to the tRNA. The mechanism of this step is
ROC CO OO PO OOCH2 somewhat different for the two classes of
A B A

NH3 O O aminoacyl-tRNA synthetases (see Table 277).
O Adenine For class I enzymes, 2a the aminoacyl group
H H is transferred initially to the 2-hydroxyl group
H H of the 3-terminal A residue, then 3a to the
5-Aminoacyl adenylate
3-hydroxyl group by a transesterification
OH OH (aminoacyl-AMP)
reaction. For class II enzymes, 2b the
class I class II aminoacyl group is transferred directly to the
aminoacyl-tRNA aminoacyl-tRNA
synthetases synthetases 3-hydroxyl group of the terminal adenylate.

3 end of tRNA

Adenine H Adenine H
2 O 2 O
H OH H B H OH H B
O ROC CO OO PO OO Adenosine O ROC CO OO PO O O Adenosine
A B A A B A
H OH  H OH
3 NH3 O O 3

NH3 O O
CH2 H CH2 H
A Aminoacyl-AMP A Aminoacyl-AMP
O O
A A
 
O OP PO O OP PO
A A
O O

tRNA tRNA

2a AMP 2b AMP

Adenine H Adenine H
2
H 2
H O COCOR H OH
B A
O O
O NH3 H
H 3
OH H 3
O COCOR
B A
CH2
A
H CH2
A
H O NH3
O transesterification
O
A A
 
O OP PO 3a O OP P O
A A
O O

Aminoacyl-tRNA
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27.2 Protein Synthesis 1053

3 end of tRNA low as that of DNA replication. Because flaws in a pro-


tein are eliminated when the protein is degraded and
Adenine H
2 are not passed on to future generations, they have less
H OH
H biological significance. The degree of fidelity in protein
O A synthesis is sufficient to ensure that most proteins con-
H OOC COR
3 B A tain no mistakes and that the large amount of energy
CH2 H 
O NH3 required to synthesize a protein is rarely wasted. One
A
O Aminoacyl defective protein molecule is usually unimportant when
A group

O OP P O many correct copies of the same protein are present.
A
O Interaction between an Aminoacyl-tRNA Synthetase and a tRNA:
A Second Genetic Code An individual aminoacyl-tRNA
5 Amino acid synthetase must be specific not only for a single amino
pG arm
acid but for certain tRNAs as well. Discriminating among
D TwC dozens of tRNAs is just as important for the overall fi-
arm arm delity of protein biosynthesis as is distinguishing among
Anticodon amino acids. The interaction between aminoacyl-tRNA
arm synthetases and tRNAs has been referred to as the sec-
ond genetic code, reflecting its critical role in main-
FIGURE 2715 General structure of aminoacyl-tRNAs. The amino-
taining the accuracy of protein synthesis. The coding
acyl group is esterified to the 3 position of the terminal A residue.
rules appear to be more complex than those in the first
The ester linkage that both activates the amino acid and joins it to the
tRNA is shaded pink.
code.
Figure 2716 summarizes what we know about the
nucleotides involved in recognition by some aminoacyl-
incorrect aminoacyl-AMP products to a separate active tRNA synthetases. Some nucleotides are conserved in all
site on the enzyme; a substrate that binds in this sec- tRNAs and therefore cannot be used for discrimination.
ond active site is hydrolyzed. The R group of valine is
slightly smaller than that of isoleucine, so Val-AMP fits 3
the hydrolytic (proofreading) site of the Ile-tRNA syn-
Amino acid
thetase but Ile-AMP does not. Thus Val-AMP is hy- arm
drolyzed to valine and AMP in the proofreading active 5
site, and tRNA bound to the synthetase does not be-
come aminoacylated to the wrong amino acid.
COO COO
 
H3N C H H3N C H TwC arm
D arm
H C CH3 H C CH3

CH3 CH2

CH3
Valine Isoleucine
Extra arm
In addition to proofreading after formation of the
aminoacyl-AMP intermediate, most aminoacyl-tRNA
synthetases can also hydrolyze the ester linkage be-
tween amino acids and tRNAs in the aminoacyl-tRNAs.
Anticodon
This hydrolysis is greatly accelerated for incorrectly arm
charged tRNAs, providing yet a third filter to enhance
the fidelity of the overall process. The few aminoacyl-
Anticodon
tRNA synthetases that activate amino acids with no
close structural relatives (Cys-tRNA synthetase, for ex- FIGURE 2716 Nucleotide positions in tRNAs that are recognized
ample) demonstrate little or no proofreading activity; in by aminoacyl-tRNA synthetases. Some positions (blue dots) are the
these cases, the active site for aminoacylation can suf- same in all tRNAs and therefore cannot be used to discriminate one
ficiently discriminate between the proper substrate and from another. Other positions are known recognition points for one
any incorrect amino acid. (orange) or more (green) aminoacyl-tRNA synthetases. Structural fea-
The overall error rate of protein synthesis (~1 mis- tures other than sequence are important for recognition by some of
take per 104 amino acids incorporated) is not nearly as the synthetases.
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1056 Chapter 27 Protein Metabolism

IF-3 IF-1
tRNA that is distinct from the tRNAMet used at (5)AUG
codons at interior positions in the mRNA. Polypeptides
synthesized by mitochondrial and chloroplast ribo-
somes, however, begin with N-formylmethionine. This
strongly supports the view that mitochondria and P A
30S
chloroplasts originated from bacterial ancestors that Subunit
were symbiotically incorporated into precursor eukary-
otic cells at an early stage of evolution (see Fig. 136). mRNA
How can the single (5)AUG codon distinguish be-
tween the starting N-formylmethionine (or methionine,
in eukaryotes) and interior Met residues? The details of 1 Initiation mRNA
the initiation process provide the answer. codon
5 A U G 3
The Three Steps of Initiation The initiation of polypep- P IF-1
IF-3
tide synthesis in bacteria requires (1) the 30S ribosomal
subunit, (2) the mRNA coding for the polypeptide to be
made, (3) the initiating fMet-tRNAf Met, (4) a set of three
proteins called initiation factors (IF-1, IF-2, and IF-3),
2 IF-2
(5) GTP, (6) the 50S ribosomal subunit, and (7) Mg2. 5
fMet
Formation of the initiation complex takes place in three
GTP
steps (Fig. 2720).
5
In step 1 the 30S ribosomal subunit binds two ini- fMet
tiation factors, IF-1 and IF-3. Factor IF-3 prevents the
C tRNA
30S and 50S subunits from combining prematurely. The U A
mRNA then binds to the 30S subunit. The initiating (3) UAC (5) IF-2 GTP
(5)AUG is guided to its correct position by the Shine- Anticodon U A C
Dalgarno sequence (named for Australian researchers
5 A U G 3
John Shine and Lynn Dalgarno, who identified it) in the
P IF-1
mRNA. This consensus sequence is an initiation signal IF-3
of four to nine purine residues, 8 to 13 bp to the 5 side
of the initiation codon (Fig. 2721a). The sequence
base-pairs with a complementary pyrimidine-rich se- 50S
quence near the 3 end of the 16S rRNA of the 30S ri- Subunit
GDP  Pi
bosomal subunit (Fig. 2721b). This mRNA-rRNA in-
teraction positions the initiating (5)AUG sequence of
the mRNA in the precise position on the 30S subunit IF-1  IF-2  IF-3
where it is required for initiation of translation. The par-
ticular (5)AUG where fMet-tRNAfMet is to be bound is
3
distinguished from other methionine codons by its prox- 5
imity to the Shine-Dalgarno sequence in the mRNA. fMet
Bacterial ribosomes have three sites that bind
aminoacyl-tRNAs, the aminoacyl (A) site, the pep-
50S
tidyl (P) site, and the exit (E) site. Both the 30S Subunit E U A C
and the 50S subunits contribute to the characteristics
of the A and P sites, whereas the E site is largely con- 5 A U G 3
fined to the 50S subunit. The initiating (5)AUG is P A Next
positioned at the P site, the only site to which fMet- codon
tRNAfMet can bind (Fig. 2720). The fMet-tRNAfMet is
the only aminoacyl-tRNA that binds first to the P site;
during the subsequent elongation stage, all other in- FIGURE 2720 Formation of the initiation complex in bacteria. The
coming aminoacyl-tRNAs (including the Met-tRNAMet complex forms in three steps (described in the text) at the expense of
that binds to interior AUG codons) bind first to the A the hydrolysis of GTP to GDP and Pi. IF-1, IF-2, and IF-3 are initia-
site and only subsequently to the P and E sites. The E tion factors. P designates the peptidyl site, A the aminoacyl site, and
site is the site from which the uncharged tRNAs leave E the exit site. Here the anticodon of the tRNA is oriented 3 to 5,
during elongation. Factor IF-1 binds at the A site and left to right, as in Figure 278 but opposite to the orientation in Fig-
prevents tRNA binding at this site during initiation. ures 2716 and 2718.
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27.2 Protein Synthesis 1057

E. coli trpA (5) A G C A C G A G G G G A A A U C U G A U G G A A C G C U A C (3)


E. coli araB U U U G G A U G G A G U G A A A C G A U G G C G A U U G C A
E. coli lacI C A A U U C A G G G U G G U G A A U G U G A A A C C A G U A
fX174 phage A protein A A U C U U G G A G G C U U U U U U A U G G U U C G U U C U
l phage cro A U G U A C U A A G G A G G U U G U A U G G A A C A A C G C

Shine-Dalgarno sequence; Initiation codon;


pairs with 16S rRNA pairs with fMet-tRNAfMet

(a)

3
OH G
3 End of A
Prokaryotic 16S rRNA A U
mRNA U C
with consensus U C C U C C A
Shine-Dalgarno
sequence (5) G A U U C C U A G G A G G U U U G A C C U A U G C G A G C U U U U A G U (3)

(b)

FIGURE 2721 Messenger RNA sequences that serve as signals for shown. Note the unusual example of the E. coli LacI protein, which
initiation of protein synthesis in bacteria. (a) Alignment of the initi- initiates with a GUG (Val) codon (see Box 272). (b) The Shine-
ating AUG (shaded in green) at its correct location on the 30S ribo- Dalgarno sequence of the mRNA pairs with a sequence near the 3
somal subunit depends in part on upstream Shine-Dalgarno sequences end of the 16S rRNA.
(pink). Portions of the mRNA transcripts of five prokaryotic genes are

In step 2 of the initiation process (Fig. 2720), the protein (PAB). Eukaryotic cells have at least nine initi-
complex consisting of the 30S ribosomal subunit, IF-3, ation factors. A complex called eIF4F, which includes
and mRNA is joined by both GTP-bound IF-2 and the the proteins eIF4E, eIF4G, and eIF4A, binds to the 5
initiating fMet-tRNAfMet. The anticodon of this tRNA cap (see Fig. 2612) through eIF4E. The protein eIF4G
now pairs correctly with the mRNAs initiation codon. binds to both eIF4E and PAB, effectively tying them to-
In step 3 this large complex combines with the 50S gether (Fig. 2722). The protein eIF4A has an RNA he-
ribosomal subunit; simultaneously, the GTP bound to licase activity. It is the eIF4F complex that associates
IF-2 is hydrolyzed to GDP and Pi, which are released
from the complex. All three initiation factors depart
from the ribosome at this point. 40S Ribosomal subunit
Completion of the steps in Figure 2720 produces
a functional 70S ribosome called the initiation com-
eIF3
plex, containing the mRNA and the initiating fMet- 5 cap 3 poly(A) tail
tRNAfMet. The correct binding of the fMet-tRNAfMet to A
the P site in the complete 70S initiation complex is as- AA A
(A) n
sured by at least three points of recognition and at-
eIF4E PAB
tachment: the codon-anticodon interaction involving the eIF4G
initiation AUG fixed in the P site; interaction between AUG
the Shine-Dalgarno sequence in the mRNA and the 16S
rRNA; and binding interactions between the ribosomal
P site and the fMet-tRNAfMet. The initiation complex is
now ready for elongation.
Gene 3 Untranslated
region
Initiation in Eukaryotic Cells Translation is generally sim-
ilar in eukaryotic and bacterial cells; most of the signif- FIGURE 2722 Protein complexes in the formation of a eukaryotic
icant differences are in the mechanism of initiation. Eu- initiation complex. The 3 and 5 ends of eukaryotic mRNAs are linked
karyotic mRNAs are bound to the ribosome as a complex by a complex of proteins that includes several initiation factors and
with a number of specific binding proteins. Several of the poly(A) binding protein (PAB). The factors eIF4E and eIF4G are
these tie together the 5 and 3 ends of the message. At part of a larger complex called eIF4F. This complex binds to the 40S
the 3 end, the mRNA is bound by the poly(A) binding ribosomal subunit.
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1058 Chapter 27 Protein Metabolism

TABLE 278 Protein Factors Required for Initiation of Translation in Bacterial and Eukaryotic Cells
Factor Function
Bacterial
IF-1 Prevents premature binding of tRNAs to A site
IF-2 Facilitates binding of fMet-tRNAfMet to 30S ribosomal subunit
IF-3 Binds to 30S subunit; prevents premature association of 50S
subunit; enhances specificity of P site for fMet-tRNAfMet
Eukaryotic*
eIF2 Facilitates binding of initiating Met-tRNAMet to 40S ribosomal subunit
eIF2B, eIF3 First factors to bind 40S subunit; facilitate subsequent steps
eIF4A RNA helicase activity removes secondary structure in the mRNA to permit binding
to 40S subunit; part of the eIF4F complex
eIF4B Binds to mRNA; facilitates scanning of mRNA to locate the first AUG
eIF4E Binds to the 5 cap of mRNA; part of the eIF4F complex
eIF4G Binds to eIF4E and to poly(A) binding protein (PAB); part of the eIF4F complex
eIF5 Promotes dissociation of several other initiation factors from 40S subunit as a
prelude to association of 60S subunit to form 80S initiation complex
eIF6 Facilitates dissociation of inactive 80S ribosome into 40S and 60S subunits

*
The prefix e identifies these as eukaryotic factors.

with another factor, eIF3, and with the 40S ribosomal Elongation Step 1: Binding of an Incoming Aminoacyl-tRNA In
subunit. The efficiency of translation is affected by many the first step of the elongation cycle (Fig. 2723), the
properties of the mRNA and proteins in this complex, appropriate incoming aminoacyl-tRNA binds to a com-
including the length of the 3 poly(A) tract (in most plex of GTP-bound EF-Tu. The resulting aminoacyl-
cases, longer is better). The end-to-end arrangement of tRNAEF-TuGTP complex binds to the A site of the
the eukaryotic mRNA facilitates translational regulation 70S initiation complex. The GTP is hydrolyzed and an
of gene expression, considered in Chapter 28. EF-TuGDP complex is released from the 70S ribosome.
The initiating (5)AUG is detected within the mRNA The EF-TuGTP complex is regenerated in a process in-
not by its proximity to a Shine-Dalgarno-like sequence volving EF-Ts and GTP.
but by a scanning process: a scan of the mRNA from the
5 end until the first AUG is encountered, signaling the Elongation Step 2: Peptide Bond Formation A peptide bond
beginning of the reading frame. The eIF4F complex is is now formed between the two amino acids bound by
probably involved in this process, perhaps using the their tRNAs to the A and P sites on the ribosome. This
RNA helicase activity of eIF4A to eliminate secondary occurs by the transfer of the initiating N-formylme-
structure in the 5 untranslated portion of the mRNA. thionyl group from its tRNA to the amino group of the
Scanning is also facilitated by another protein, eIF4B. second amino acid, now in the A site (Fig. 2724). The
The roles of the various bacterial and eukaryotic ini- -amino group of the amino acid in the A site acts as a
tiation factors in the overall process are summarized in nucleophile, displacing the tRNA in the P site to form
Table 278. The mechanism by which these proteins act the peptide bond. This reaction produces a dipeptidyl-
is an important area of investigation. tRNA in the A site, and the now uncharged (deacy-
lated) tRNAfMet remains bound to the P site. The tRNAs
Stage 3: Peptide Bonds Are Formed in the then shift to a hybrid binding state, with elements of
each spanning two different sites on the ribosome, as
Elongation Stage
shown in Figure 2724.
The third stage of protein synthesis is elongation. The enzymatic activity that catalyzes peptide bond
Again, our initial focus is on bacterial cells. Elongation formation has historically been referred to as peptidyl
requires (1) the initiation complex described above, (2) transferase and was widely assumed to be intrinsic to
aminoacyl-tRNAs, (3) a set of three soluble cytosolic one or more of the proteins in the large ribosomal sub-
proteins called elongation factors (EF-Tu, EF-Ts, and unit. We now know that this reaction is catalyzed by the
EF-G in bacteria), and (4) GTP. Cells use three steps to 23S rRNA (Fig. 279), adding to the known catalytic
add each amino acid residue, and the steps are repeated repertoire of ribozymes. This discovery has interesting
as many times as there are residues to be added. implications for the evolution of life (Box 273).
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27.2 Protein Synthesis 1059

Initiation 5
fMet
complex

50S U A C
E E site P site A site
5 A U G 3

Initiation P A Next
codon codon
H fMet-tRNAfMet
30S 5
AA2 C O
.. Aminoacyl-
Incoming NH NH2 tRNA2
aminoacyl- R1 C H R2 C H
tRNA
O C O C
O O
5 5
5 AA
2

Tu GTP
E U A C

Tu GTP mRNA 5 A U G 3
P A

Ts
binding of
incoming
aminoacyl-
tRNA GTP
peptide bond
formation
Tu Ts

E site P site A site


GDP
H
Pi Tu O
C
Ts
GDP NH
H Deacylated
C
R
1
tRNAfMet
C
O
NH
H
2 C
OH R C
Dipeptidyl-
5 5 AA O
O tRNA2
fMet 2
5 5 5 5

E U A C
E U A C
5 A U G 3 5 3
A U G
P A P A

FIGURE 2723 First elongation step in bacteria: binding of the sec- FIGURE 2724 Second elongation step in bacteria: formation of the
ond aminoacyl-tRNA. The second aminoacyl-tRNA enters the A site first peptide bond. The peptidyl transferase catalyzing this reaction is
of the ribosome bound to EF-Tu (shown here as Tu), which also con- the 23S rRNA ribozyme. The N-formylmethionyl group is transferred
tains GTP. Binding of the second aminoacyl-tRNA to the A site is ac- to the amino group of the second aminoacyl-tRNA in the A site, form-
companied by hydrolysis of the GTP to GDP and Pi and release of the ing a dipeptidyl-tRNA. At this stage, both tRNAs bound to the ribo-
EF-TuGDP complex from the ribosome. The bound GDP is released some shift position in the 50S subunit to take up a hybrid binding
when the EF-TuGDP complex binds to EF-Ts, and EF-Ts is subse- state. The uncharged tRNA shifts so that its 3 and 5 ends are in the
quently released when another molecule of GTP binds to EF-Tu. This E site. Similarly, the 3 and 5 ends of the peptidyl tRNA shift to the
recycles EF-Tu and makes it available to repeat the cycle. P site. The anticodons remain in the A and P sites.
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1060 Chapter 27 Protein Metabolism

Elongation Step 3: Translocation In the final step of the A change in the three-dimensional conformation of the
elongation cycle, translocation, the ribosome moves entire ribosome results in its movement along the mRNA.
one codon toward the 3 end of the mRNA (Fig. 2725a). Because the structure of EF-G mimics the structure of
This movement shifts the anticodon of the dipeptidyl- the EF-TutRNA complex (Fig. 2725b), EF-G can bind
tRNA, which is still attached to the second codon of the the A site and presumably displace the peptidyl-tRNA.
mRNA, from the A site to the P site, and shifts the de-
acylated tRNA from the P site to the E site, from where The ribosome, with its attached dipeptidyl-tRNA and
the tRNA is released into the cytosol. The third codon mRNA, is now ready for the next elongation cycle and
of the mRNA now lies in the A site and the second codon attachment of a third amino acid residue. This process
in the P site. Movement of the ribosome along the mRNA occurs in the same way as addition of the second residue
requires EF-G (also known as translocase) and the en- (as shown in Figs 2723, 2724, and 2725). For each
ergy provided by hydrolysis of another molecule of GTP. amino acid residue correctly added to the growing
polypeptide, two GTPs are hydrolyzed to GDP and Pi as
the ribosome moves from codon to codon along the
E site P site A site
mRNA toward the 3 end.
H
The polypeptide remains attached to the tRNA of
O the most recent amino acid to be inserted. This associ-
C
NH ation maintains the functional connection between the
H Deacylated
1 C
tRNAfMet
information in the mRNA and its decoded polypeptide
R
C
O output. At the same time, the ester linkage between this
NH tRNA and the carboxyl terminus of the growing polypep-
H Dipeptidyl-
2 C
OH R C tRNA2 tide activates the terminal carboxyl group for nucleo-
O
O philic attack by the incoming amino acid to form a new
5 5 5 5 peptide bond (Fig. 2724). As the existing ester linkage
between the polypeptide and tRNA is broken during

E U A C
FIGURE 2725 Third elongation step in bacteria: translocation.
5 A U G 3
(a) The ribosome moves one codon toward the 3 end of the mRNA,
P A
using energy provided by hydrolysis of GTP bound to EF-G (translo-
case). The dipeptidyl-tRNA is now entirely in the P site, leaving the A
site open for the incoming (third) aminoacyl-tRNA. The uncharged
tRNA dissociates from the E site, and the elongation cycle begins again.
(b) The structure of EF-G mimics the structure of EF-Tu complexed with
EF-G GTP
tRNA. Shown here are (left) EF-Tu complexed with tRNA (green) (PDB
translocation ID 1B23) and (right) EF-G complexed with GDP (red) (PDB ID 1DAR).
EF-G  GDP  Pi The carboxyl-terminal part of EF-G (dark gray) mimics the structure of
the anticodon loop of tRNA in both shape and charge distribution.

E site P site A site


H
C O
NH
1 C H
R Incoming
O C aminoacyl-tRNA3
NH
5
2 C H
R
O C
OH
O
5 5 5 5

U A C
5 A U G 3
P A

Direction of
ribosome movement
(a) (b)
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1062 Chapter 27 Protein Metabolism

is a single molecule of mRNA that is being translated si-


multaneously by many closely spaced ribosomes, allow-
Release factor ing the highly efficient use of the mRNA.
5 binds In bacteria, transcription and translation are tightly
RF
coupled. Messenger RNAs are synthesized and trans-
lated in the same 5n 3 direction. Ribosomes begin
translating the 5 end of the mRNA before transcription
E is complete (Fig. 2728). The situation is quite differ-
5 U A G 3 ent in eukaryotic cells, where newly transcribed mRNAs
P A must leave the nucleus before they can be translated.
Bacterial mRNAs generally exist for just a few min-
utes (p. 1020) before they are degraded by nucleases.
polypeptidyl-tRNA In order to maintain high rates of protein synthesis, the
link hydrolyzed
mRNA for a given protein or set of proteins must be
made continuously and translated with maximum effi-
ciency. The short lifetime of mRNAs in bacteria allows
COO
a rapid cessation of synthesis when the protein is no
5 longer needed.

Stage 5: Newly Synthesized Polypeptide Chains


RF Undergo Folding and Processing
E
5 U A G 3 In the final stage of protein synthesis, the nascent
P A
polypeptide chain is folded and processed into its bio-
logically active form. During or after its synthesis, the
polypeptide progressively assumes its native conforma-
tion, with the formation of appropriate hydrogen bonds
and van der Waals, ionic, and hydrophobic interactions.
components
dissociate In this way the linear, or one-dimensional, genetic
message in the mRNA is converted into the three-
5
dimensional structure of the protein. Some newly made
proteins, both prokaryotic and eukaryotic, do not attain
their final biologically active conformation until they
RF
have been altered by one or more processing reactions
called posttranslational modifications.
5 U A G 3
Amino-Terminal and Carboxyl-Terminal Modifications The first
residue inserted in all polypeptides is N-formylmethio-
nine (in bacteria) or methionine (in eukaryotes). How-
ever, the formyl group, the amino-terminal Met residue,
and often additional amino-terminal (and, in some cases,
FIGURE 2726 Termination of protein synthesis in bacteria. Termi- carboxyl-terminal) residues may be removed enzymat-
nation occurs in response to a termination codon in the A site. First, ically in formation of the final functional protein. In as
a release factor, RF (RF-1 or RF-2, depending on which termination many as 50% of eukaryotic proteins, the amino group
codon is present), binds to the A site. This leads to hydrolysis of the
of the amino-terminal residue is N-acetylated after
ester linkage between the nascent polypeptide and the tRNA in the P
translation. Carboxyl-terminal residues are also some-
site and release of the completed polypeptide. Finally, the mRNA, de-
times modified.
acylated tRNA, and release factor leave the ribosome, and the ribo-
some dissociates into its 30S and 50S subunits. Loss of Signal Sequences As we shall see in Section 27.3,
the 15 to 30 residues at the amino-terminal end of some
proteins play a role in directing the protein to its ulti-
Rapid Translation of a Single Message by Polysomes Large mate destination in the cell. Such signal sequences are
clusters of 10 to 100 ribosomes that are very active in ultimately removed by specific peptidases.
protein synthesis can be isolated from both eukaryotic
and bacterial cells. Electron micrographs show a fiber Modification of Individual Amino Acids The hydroxyl
between adjacent ribosomes in the cluster, which is groups of certain Ser, Thr, and Tyr residues of some pro-
called a polysome (Fig. 2727). The connecting strand teins are enzymatically phosphorylated by ATP (Fig.
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27.2 Protein Synthesis 1063

0.25 mm

(b)

FIGURE 2727 Polysome. (a) Four ribosomes translating a eukaryotic


mRNA molecule simultaneously, moving from the 5 end to the 3
end and synthesizing a polypeptide from the amino terminus to the
carboxyl terminus. (b) Electron micrograph and explanatory diagram
of a polysome from the silk gland of a silkworm larva. The mRNA is
being translated by many ribosomes simultaneously. The nascent
polypeptides become longer as the ribosomes move toward the 3 end
of the mRNA. The final product of this process is silk fibroin.

2729a); the phosphate groups add negative charges to amino acids are all valuable to suckling young, so casein
these polypeptides. The functional significance of this efficiently provides three essential nutrients. And as we
modification varies from one protein to the next. For have seen in numerous instances, phosphorylation-
example, the milk protein casein has many phospho- dephosphorylation cycles regulate the activity of many
serine groups that bind Ca2. Calcium, phosphate, and enzymes and regulatory proteins.
Extra carboxyl groups may be added to Glu residues
of some proteins. For example, the blood-clotting pro-
DNA RNA
tein prothrombin contains a number of -carboxygluta-
3 mate residues (Fig. 2729b) in its amino-terminal re-
5 duplex polymerase 5
3 gion, introduced by an enzyme that requires vitamin K.
These carboxyl groups bind Ca2, which is required to
initiate the clotting mechanism.

Ribosome
FIGURE 2728 Coupling of transcription and translation in bacte-
5 ria. The mRNA is translated by ribosomes while it is still being tran-
mRNA scribed from DNA by RNA polymerase. This is possible because the
Direction of transcription mRNA in bacteria does not have to be transported from a nucleus to

 NH3 the cytoplasm before encountering ribosomes. In this schematic dia-
NH3
gram the ribosomes are depicted as smaller than the RNA polymerase.
Direction of translation
In reality the ribosomes (Mr 2.7  106) are an order of magnitude
larger than the RNA polymerase (Mr 3.9  105).
8885d_c27_1034-1080 2/12/04 1:19 PM Page 1066 mac76 mac76:385_reb:

1066 Chapter 27 Protein Metabolism


..

P site A site
peptidyl-tRNA puromycin

CH2 OCH3
..
H2N C H
NH
C O
R C H
NH OH
C O H H
H H
O OH CH O2
H H
H H HO N N
CH O2 N N
O N N N
CH3 CH3
P N N
5 NH2

AA
5
NH
R C H CH2 OCH3
O C N C H
E H
5 C O
3
mRNA P A NH OH
H H
H H
CH O2
peptidyl N
HO N
transferase
N N
N
CH3 CH3
5

(b)

FIGURE 2731 Disruption of peptide bond formation by puromycin.


E
5 3 (a) The antibiotic puromycin resembles the aminoacyl end of a charged
P A tRNA, and it can bind to the ribosomal A site and participate in pep-
tide bond formation. The product of this reaction, instead of being
translocated to the P site, dissociates from the ribosome, causing pre-
(a) mature chain termination. (b) Peptidyl puromycin.

Puromycin, made by the mold Streptomyces al-


boniger, is one of the best-understood inhibitory an-
CH3 CH3
tibiotics. Its structure is very similar to the 3 end of an
CH3 OH N
aminoacyl-tRNA, enabling it to bind to the ribosomal A H OH
site and participate in peptide bond formation, produc-
ing peptidyl-puromycin (Fig. 2731). However, because
puromycin resembles only the 3 end of the tRNA, it OH CONH2
does not engage in translocation and dissociates from OH O OH O
the ribosome shortly after it is linked to the carboxyl Tetracycline
terminus of the peptide. This prematurely terminates NH C CHCl2
polypeptide synthesis. O2N CH CH O
Tetracyclines inhibit protein synthesis in bacteria
OH CH2
by blocking the A site on the ribosome, preventing the CH3
binding of aminoacyl-tRNAs. Chloramphenicol in- OH
hibits protein synthesis by bacterial (and mitochondrial H 3C O Chloramphenicol
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27.3 Protein Targeting and Degradation 1069

5 cap
Signal
sequence
2 1
mRNA
GUA

8
Ribosome
cycle
SRP SRP
Cytosol
cycle )n
A
3 A(
AA
5 GDP  Pi

Ribosome
receptor GTP 4 6

Peptide SRP Signal 7


translocation receptor peptidase
Endoplasmic complex
reticulum

ER lumen

FIGURE 2733 Directing eukaryotic proteins with the appropriate the signal sequence, inhibiting elongation by sterically blocking the
signals to the endoplasmic reticulum. This process involves the SRP entry of aminoacyl-tRNAs and inhibiting peptidyl transferase. Another
cycle and translocation and cleavage of the nascent polypeptide. The protein subunit binds and hydrolyzes GTP. The SRP receptor is a het-
steps are described in the text. SRP is a rod-shaped complex con- erodimer of  (Mr 69,000) and  (Mr 30,000) subunits, both of which
taining a 300 nucleotide RNA (7SL-RNA) and six different proteins bind and hydrolyze multiple GTP molecules during this process.
(combined Mr 325,000). One protein subunit of SRP binds directly to

steps 1 through 8 in Figure 2733. 1 The targeting Glycosylation Plays a Key Role in Protein Targeting
pathway begins with initiation of protein synthesis on
In the ER lumen, newly synthesized proteins are further
free ribosomes. 2 The signal sequence appears early in
modified in several ways. Following the removal of sig-
the synthetic process, because it is at the amino termi-
nal sequences, polypeptides are folded, disulfide bonds
nus, which as we have seen is synthesized first. 3 As it
formed, and many proteins glycosylated to form glyco-
emerges from the ribosome, the signal sequenceand
proteins. In many glycoproteins the linkage to their
the ribosome itselfare bound by the large signal
oligosaccharides is through Asn residues. These N-
recognition particle (SRP); SRP then binds GTP and
linked oligosaccharides are diverse (Chapter 7), but the
halts elongation of the polypeptide when it is about 70
pathways by which they form have a common first step.
amino acids long and the signal sequence has completely
A 14 residue core oligosaccharide is built up in a step-
emerged from the ribosome. 4 The GTP-bound SRP
wise fashion, then transferred from a dolichol phosphate
now directs the ribosome (still bound to the mRNA) and
donor molecule to certain Asn residues in the protein
the incomplete polypeptide to GTP-bound SRP recep-
(Fig. 2734). The transferase is on the lumenal face of
tors in the cytosolic face of the ER; the nascent polypep-
the ER and thus cannot catalyze glycosylation of cyto-
tide is delivered to a peptide translocation complex
solic proteins. After transfer, the core oligosaccharide is
in the ER, which may interact directly with the ribo-
trimmed and elaborated in different ways on different
some. 5 SRP dissociates from the ribosome, accompa-
nied by hydrolysis of GTP in both SRP and the SRP re-
ceptor. 6 Elongation of the polypeptide now resumes,
CH3 CH3
with the ATP-driven translocation complex feeding the
growing polypeptide into the ER lumen until the com- P CH3
plete protein has been synthesized. 7 The signal se- CH3 n

quence is removed by a signal peptidase within the ER Dolichol phosphate


lumen; 8 the ribosome dissociates and is recycled. (n  922)
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1070 Chapter 27 Protein Metabolism


N-Acetylglucosamine (GlcNAc)
tunicamycin Mannose (Man)
Glucose (Glc)
5 GDP 5 GDP-Man UMP  UDP 2 UDP-GlcNAc

P P
translocation
P 2 P 1 P dolichol
3 phosphate
recycled

Dolichol P Pi
Endoplasmic
P
P

reticulum 9

H 3N
4 Dolichol P Man
4 Dolichol P  P P
 H3N
3 Dolichol P Glc NH3
3 Dolichol P
4 P P
8
P 5 Asn 6 7 P

Cytosol
5
mRNA
3

FIGURE 2734 Synthesis of the core oligosaccharide of glycopro- oligosaccharide is transferred from dolichol phosphate to an Asn
teins. The core oligosaccharide is built up by the successive addition residue of the protein within the ER lumen. The core oligosaccharide
of monosaccharide units. 1 , 2 The first steps occur on the cytoso- is then further modified in the ER and the Golgi complex in pathways
lic face of the ER. 3 Translocation moves the incomplete oligosac- that differ for different proteins. The five sugar residues shown sur-
charide across the membrane (mechanism not shown), and 4 com- rounded by a beige screen (after step 7 ) are retained in the final
pletion of the core oligosaccharide occurs within the lumen of the ER. structure of all N-linked oligosaccharides. 8 The released dolichol
The precursors that contribute additional mannose and glucose pyrophosphate is again translocated so that the pyrophosphate is on
residues to the growing oligosaccharide in the lumen are dolichol the cytosolic face of the ER, then 9 a phosphate is hydrolytically re-
phosphate derivatives. In the first step in the construction of the N- moved to regenerate dolichol phosphate.
linked oligosaccharide moiety of a glycoprotein, 5 , 6 the core

proteins, but all N-linked oligosaccharides retain a pen- CH2OH


tasaccharide core derived from the original 14 residue O Tunicamycin
oligosaccharide. Several antibiotics act by interfering H H
H
with one or more steps in this process and have aided OH H
in elucidating the steps of protein glycosylation. The HO
N-Acetylglucosamine
best-characterized is tunicamycin, which mimics the
H NH
structure of UDP-N-acetylglucosamine and blocks the A
first step of the process (Fig. 2734, step 1 ). A few pro- O C
A
teins are O-glycosylated in the ER, but most O-glyco- CH3
sylation occurs in the Golgi complex or in the cytosol
O O
(for proteins that do not enter the ER).
O
Suitably modified proteins can now be moved to a H H HN
H
variety of intracellular destinations. Proteins travel from Uracil
OH H
the ER to the Golgi complex in transport vesicles (Fig. HN CH2 O N
2735). In the Golgi complex, oligosaccharides are O- A
linked to some proteins, and N-linked oligosaccharides H OH CHOH
O C O
are further modified. By mechanisms not yet fully un-
derstood, the Golgi complex also sorts proteins and H Ca H H
sends them to their final destinations. The processes H H
H Cb
that segregate proteins targeted for secretion from Fatty acyl OH OH
those targeted for the plasma membrane or lysosomes side chain (CH2)n
Tunicamine
must distinguish among these proteins on the basis of CH
structural features other than signal sequences, which CH3 CH3
were removed in the ER lumen. (n  8 11)
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1072 Chapter 27 Protein Metabolism

CH2OH CH2OH
O O
H H H H
H H
O O 
OH H B B OH HO H
HO OO PO OO PO O O Uridine HO OO Oligosaccharide O NO Enzyme
A A
H NH O O H H Hydrolase
A
CPO Mannose residue
A
CH3
UDP N-Acetylglucosamine
(UDP-GlcNAc) N-acetylglucosamine
phosphotransferase UMP

CH2OH
O
H H H
O
OH H B
HO O OP OO OCH2
A
H NH O O
A
H H H
CPO
A
CH3 OH HO H
HO OO Oligosaccharide O NO Enzyme
H H

phosphodiesterase GlcNAc

O
B

O OP OO OCH2
A
O
O
H H
H
OH HO H
FIGURE 2736 Phosphorylation of mannose residues HO OO Oligosaccharide O NO Enzyme
on lysosome-targeted enzymes. N-Acetylglucosamine
phosphotransferase recognizes some as yet unidentified H H
structural feature of hydrolases destined for lysosomes. Mannose 6-phosphate residue

proteins in the cytosol. The complex of the NLS- 2738), much like those on eukaryotic proteins targeted
bearing protein and the importin docks at a nuclear pore to the ER, mitochondria, and chloroplasts.
and is translocated through the pore by an energy- Most proteins exported from E. coli make use of
dependent mechanism that requires the Ran GTPase. the pathway shown in Figure 2739. Following transla-
The two importin subunits separate during the translo- tion, a protein to be exported may fold only slowly, the
cation, and the NLS-bearing protein dissociates from im- amino-terminal signal sequence impeding the folding.
portin  inside the nucleus. Importin  and  are then The soluble chaperone protein SecB binds to the pro-
exported from the nucleus to repeat the process. How teins signal sequence or other features of its incom-
importin  remains dissociated from the many NLS- pletely folded structure. The bound protein is then de-
bearing proteins inside the nucleus is not yet clear. livered to SecA, a protein associated with the inner
surface of the plasma membrane. SecA acts as both a
Bacteria Also Use Signal Sequences receptor and a translocating ATPase. Released from
SecB and bound to SecA, the protein is delivered to a
for Protein Targeting
translocation complex in the membrane, made up of
Bacteria can target proteins to their inner or outer mem- SecY, E, and G, and is translocated stepwise through the
branes, to the periplasmic space between these mem- membrane at the SecYEG complex in lengths of about
branes, or to the extracellular medium. They use signal 20 amino acid residues. Each step is facilitated by the
sequences at the amino terminus of the proteins (Fig. hydrolysis of ATP, catalyzed by SecA.
8885d_c27_1034-1080 2/12/04 1:19 PM Page 1073 mac76 mac76:385_reb:

27.3 Protein Targeting and Degradation 1073

(a)

Nuclear
protein Cytosol

Nuclear
NLS envelope
2
1 (b)
a
Importin b

Ran
6 Nuclear 3 GTP GDP  Pi
pore
complex
4

5 a

Nucleoplasm

FIGURE 2737 Targeting of nuclear proteins. (a) 1 A protein with an appropriate nuclear 0.2 m
localization signal (NLS) is bound by a complex of importin  and . 2 The resulting complex
binds to a nuclear pore, and 3 translocation is mediated by the Ran GTPase. 4 Inside the
nucleus, importin  dissociates from importin , and 5 importin  then releases the nuclear
protein. 6 Importin  and  are transported out of the nucleus and recycled. (b) Scanning
electron micrograph of the surface of the nuclear envelope, showing numerous nuclear pores.

Inner membrane proteins


cleavage
Phage fd, major site
coat protein Met Lys Lys Ser Leu Val Leu Lys Ala Ser Val Ala Val Ala Thr Leu Val Pro Met Leu Ser Phe Ala Ala Glu
Phage fd, minor
coat protein Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser His Ser Ala Glu
Periplasmic proteins
Alkaline phosphatase Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr Pro Val Thr Lys Ala Arg Thr
Leucine-specific
binding protein Met Lys Ala Asn Ala Lys Thr Ile Ile Ala Gly Met Ile Ala Leu Ala Ile Ser His Thr Ala Met Ala Asp Asp

-Lactamase of
pBR322 Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro
Outer membrane proteins
Lipoprotein Met Lys Ala Thr Lys Leu Val Leu Gly Ala Val Ile Leu Gly Ser Thr Leu Leu Ala Gly Cys Ser
LamB Leu Arg Lys Leu Pro Leu Ala Val Ala Val Ala Ala Gly Val Met Ser Ala Gln Ala Met Ala Val Asp
OmpA Met Met Ile Thr Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala Thr Val Ala Gln Ala Ala Pro

FIGURE 2738 Signal sequences that target proteins to different lo- by red arrows. Note that the inner bacterial cell membrane (see Fig.
cations in bacteria. Basic amino acids (blue) near the amino termi- 16) is where phage fd coat proteins and DNA are assembled into
nus and hydrophobic core amino acids (yellow) are highlighted. The phage particles. OmpA is outer membrane protein A; LamB is a cell
cleavage sites marking the ends of the signal sequences are indicated surface receptor protein for bacteriophage lambda.
8885d_c27_1034-1080 2/12/04 1:19 PM Page 1074 mac76 mac76:385_reb:

1074 Chapter 27 Protein Metabolism

FIGURE 2739 Model for protein export in


bacteria. 1 A newly translated polypeptide binds
 SecB to the cytosolic chaperone protein SecB, which 2
delivers it to SecA, a protein associated with the
1 translocation complex (SecYEG) in the bacterial cell
membrane. 3 SecB is released, and SecA inserts
itself into the membrane, forcing about 20 amino
Cytosol acid residues of the protein to be exported through
2 the translocation complex. 4 Hydrolysis of an ATP
SecB by SecA provides the energy for a conformational
ATP ADP  Pi ATP change that causes SecA to withdraw from the
 SecA membrane, releasing the polypeptide. 5 SecA
binds another ATP, and the next stretch of 20 amino
 acid residues is pushed across the membrane
SecYEG 3 4 5 through the translocation complex. Steps 4 and
6 5 are repeated until 6 the entire protein has
passed through and is released to the periplasm.
Periplasmic The electrochemical potential across the membrane
space
(denoted by  and ) also provides some of the
driving force required for protein translocation.

An exported protein is thus pushed through the Cells Import Proteins by Receptor-Mediated
membrane by a SecA protein located on the cytoplas-
Endocytosis
mic surface, rather than being pulled through the mem-
brane by a protein on the periplasmic surface. This dif- Some proteins are imported into cells from the sur-
ference may simply reflect the need for the translocating rounding medium; examples in eukaryotes include low-
ATPase to be where the ATP is. The transmembrane density lipoprotein (LDL), the iron-carrying protein
electrochemical potential can also provide energy for transferrin, peptide hormones, and circulating proteins
translocation of the protein, by an as yet unknown destined for degradation. The proteins bind to recep-
mechanism. tors in invaginations of the membrane called coated
Although most exported bacterial proteins use this pits, which concentrate endocytic receptors in prefer-
pathway, some follow an alternative pathway that uses ence to other cell-surface proteins. The pits are coated
signal recognition and receptor proteins homologous to on their cytosolic side with a lattice of the protein
components of the eukaryotic SRP and SRP receptor clathrin, which forms closed polyhedral structures
(Fig. 2733). (Fig. 2740). The clathrin lattice grows as more recep-

Heavy
chain
Light ~80
chain nm

(a) (b) (c) 0.1 mm

FIGURE 2740 Clathrin. (a) Three light (L) chains (Mr 35,000) and tend to assemble into polyhedral lattices. (c) Electron micrograph
three heavy (H) chains (Mr 180,000) of the (HL)3 clathrin unit, or- of a coated pit on the cytosolic face of the plasma membrane of a
ganized as a three-legged structure called a triskelion. (b) Triskelions fibroblast.

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