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13.Translation and Proteins
13.1 Translation: Components
Essential to Protein Synthesis
• Ribosomal Structure
• tRNA structure
• Charging tRNA
Ribosomal Structure
• Because of its essential role in the expression of
genetic information, the ribosome has been
extensively analyzed.
• One bacterial cell about 10,000 ribosome
• One eukaryotic cell contains many times more.
Electron microscopy reveals that the bacterial
ribosome is about 250 um at its largest diameter
two subunits, one large and one small.
• Both subunits consist of one or more molecules of
rRNA and an array of ribosomal proteins.
The specific differences between prokaryotic and eukaryotic
ribosomes are summarized in Figure 13-1
16S rRNA
小亚基30S 930kD
70S 2520kD
21种蛋白质
大亚基50S 1590kD 23S rRNA+5S rRNA
34种蛋白质
18S rRNA
小亚基40S kD
80S 4420kD
大亚基60S kD
33种蛋白质
28S rRNA+5.8S rRNA+5rRNS
45种蛋白质
• Redundancy of the genes coding for tRNA
• Bacterial genome: 3 copies –primary transcript-30sRNA—enzymatically cleaved into three
components---23S/16S/5S
• Eukayotes: rRNA gene called rDNA, many
copies, 20/drosophila
500/ X. laevis tandem repeats separated by
spacer DNA
45S---28s/18/4.5s
In human ,these gene clusters have been localized
at near end of 13.14.15.21.22 chromosomes
5s rDNA
tRNA structure
•
•
•
•
•
•
•
tRNA占17-18%，73-90nt，
1963, Holley first tRNA structure，250species。
tRNA：
single strand 75-90tMW25000、4S
unusual, rare, or odd, 7-15 modified bases
 5´ phosophatation ，pG, 3 ´CCA-OH。
tRNA half of bases helix 4arms and 3loops，
cloverleaf model。
1-甲基次黄(嘌呤核)苷酸
次黄(嘌呤核)苷酸
NN-二甲基鸟苷酸
1-甲基鸟苷酸
假尿嘧啶核酸
-Gp
pCpCpA-
tRNA functional sites
 anticodon
 aa attachment site
 additional arms
assist binding to
ribosome
3-D structure
L-shape folded cloverleaf... but all unique
recognized by specific
aa synthetases
unique anticodons
tRNA genes
Charging tRNA
(Biochemistry)
• Before translation can proceed, the tRNA
molecules must be chemically linked to
their respective amino acids. This
activation process, called charging occurs
under the direction of enzymes called
aminoacyl tRNA synthetases .
 summary of chemical reactions
synthetase1
1. aa1 + tRNA1
aa1tRNA1
peptidyl transferase (on ribosome)
2. aa1tRNA1 + aa2tRNA2
aa1aa2tRNA2 + tRNA1
peptidyl transferase (on ribosome)
3. aa1aa2tRNA2 + aa3tRNA3
aa1aa2aa3tRNA3 + tRNA2 ...
 summary of chemical reactions
synthetase1
1. aa1 + tRNA1
aa1tRNA1
peptidyl transferase (on ribosome)
2. aa1tRNA1 + aa2tRNA2
aa1aa2tRNA2 + tRNA1
peptidyl transferase (on ribosome)
3. aa1aa2tRNA2 + aa3tRNA3
polypepti
aa1aa2aa3tRNA3 + tRNA2 ...
de
13.2 Translation process—
Protein synthesis
•
•
•
•
Initiation
Elongaton
Termination
polyribosomes
The small ribosomal subunit binds to
several initiation factors, and this
complex in turn binds to mRNA (step 1).
In bacteria, this binding involves a
sequence of up to six ribonucleotides
(AGGAGG, not shown), which precedes
the initial AUG start codon of mRNA.
This sequence (containing only purines
and called the Shine-Dalgarno sequence)
base-pairs with a region of the 16S
rRNA of the small ribosomal subunit,
facilitating initiation.
The increase of the growing polypeptide
chain by one amino acid is called
elongation. The sequence of the second
triplet in mRNA dictates which charged
tRNA molecule will become positioned at
the A site (step 1). Once it is present,
peptidyl transferase catalyzes the
formation of the pep-tide bond, which links
the two amino acids (step 2). This enzyme
is part of the large subunit of the ribosome.
At the same time, the covalent bond
between the amino acid and the tRNA
occupying the P site is hydrolyzed (broken).
The product of this reaction is a dipeptide,
which is attached to the 3;-end of tRNA
still residing in the A site.
UAG, UAA, or UGA. These
codons do not specify an amino
acid, nor do they call for a tRNA
in the A site. These codons are
called stop codons,
termination codons, or
nonsense codons. The finished
polypeptide is therefore still
attached to the terminal tRNA at
the P site, and the A site is empty.
The termination codon signals
the action of GTP-dependent
release factors, which cleave
the polypeptide chain from the
terminal tRNA, releasing it from
the translation complex (step I).
PROTEIN SYNTHESIS
 polypeptide synthesis on ribosomes (prokaryote e.g.)
PROTEIN SYNTHESIS
 mRNA binds to 30S subunit
PROTEIN SYNTHESIS
 tRNAs bind at 2 sites on the ribosome
PROTEIN SYNTHESIS
 A site = entry site for arriving aatRNAs
PROTEIN SYNTHESIS
 P site = position of growing aaaatRNA chain
PROTEIN SYNTHESIS
 peptidyl transferase-catalysis of peptide bonds
PROTEIN SYNTHESIS
 deacylated tRNA leaves P-site
PROTEIN SYNTHESIS
 ribosome moves 1 codon in 3' direction on mRNA
PROTEIN SYNTHESIS
 A-site left vacant for arriving aatRNAs
PROTEIN SYNTHESIS
 3 distinct stages of translation... protein synthesis
1. initiation
2. elongation
3. termination
PROTEIN SYNTHESIS
 initiation
 initiation factors IF1, IF2, IF3
 1st aa = modified MET... N-formylmethionine (fMET)
PROTEIN SYNTHESIS
 initiation
 initiation factors IF1, IF2, IF3
 1st aa = modified MET... N-formylmethionine (fMET)
 inserted by initiator tRNA... tRNAfMET
 recognizes start codon... AUG
 fMET start only... MET anywhere else
PROTEIN SYNTHESIS
 initiation
 30S + IF3... + mRNA =
initiation complex
 IF2 + GTP +
fMETtRNAfMET ...  P site
on mRNA
 IF2 & GTP split, ribosome
assembled, IF2 & IF3
released
PROTEIN SYNTHESIS
 initiation
 MET or fMET anticodons  AUG (GUG) codons ?
 start codon preceded by Shine-Delgarno sequence...
PROTEIN SYNTHESIS
 initiation
 MET or fMET anticodons  AUG (GUG) codons ?
 start codon preceded by Shine-Delgarno sequence...
pairs with 16S rRNA
PROTEIN
SYNTHESIS
 elongation
 elongation
factors EF-Tu,
EF-Ts, EF-G
PROTEIN SYNTHESIS
 elongation
 EF-Tu + GTP + aatRNA ...  A site
 EF-Ts mediates release/recycle of EF-Tu
PROTEIN SYNTHESIS
 elongation
 aaaa  aatRNA on A site by peptidyl transferase
 EF-G + GTP mediate ribosome translocation...
PROTEIN SYNTHESIS
 elongation
 EF-G + GTP mediate ribosome translocation 5'  3'
 tRNA released from P site
 aaaatRNA on A site  P site
PROTEIN SYNTHESIS
 termination
 release factors RF1, RF2, RF3
 RF1 & 2 recognize A site STOP
RF1 ... UAA & UAG
RF2 ... UAA & UGA
 aaaa released from P site
 ribosome dissociates
Process
Initiation
Elongation
Factor
Role
IF1
IF2
Stabilizes 305 subunit
Binds fmet-tRNA to 30S-mRNA complex;
binds to GTF and stimulate
IF3
Binds 305 subunit to mRNA
EF-Tu
EF-Ts
Binds GTP; Brings aminoacyl-tRNA to the A
site of ribosome
Generates active EF-Tu
Stimulates translocation; GTP-dependent
EF-G
Terminate
RFl
release
RF2
RF3
Catalyzes release of the polypeptide chain from tRNA an
disaociation
complex;like
Behaves
specific
RPl; for
Specific
UAA for
andUGA
UAGand
termination
UAA
codons
codons
Stimulates RF1 and RF2
PROTEIN SYNTHESIS
 how do mutations  codon function ? ...
sense   (or functional) aa
missense   aa
nonsense  STOP... no aa
PROTEIN SYNTHESIS
 Brenner, nonsense suppressor mutations in T4 phage
 normal STOP sequence
PROTEIN SYNTHESIS
 Brenner, nonsense suppressor mutations in T4 phage
 mutate anticodon sequence in tRNATyr
Polyribosome
As elongation proceeds and the initial portion of mRNA has
passed through the ribosome, this mRNA is free to associate
with another small subunit to form a second initiation complex.
This process can be repeated several times with a single
mRNA and results in what are called polyribosomes or just
polysomes.
13.3 Translation in Eukaryotes
• The general features of the model we just
discussed were initially derived from
investigations of the translation process in
bacteria. As we saw, one main difference
between translation in prokaryotes and
eukaryotes is that in the latter, translation
occurs on ribosomes that are larger and
whose rRNA and protein components are
more complex than those of prokaryotes (see
Figure 13-1).
• Several other differences are also
important. Eukaryotic mRNAs are much
longer-lived than their prokaryotic
counterparts. Most exist for hours rather
than minutes prior to their degradation
by nucleases in the cell, remaining
available much longer to orchestrate
protein synthesis.
• Several aspects that involve the initiation of
translation are different in eukaryotes.
• First, the 5'-end is "capped" with a 7methylguanosine residue. The presence of this
"cap" absent in prokaryotes. is essential to
efficient translation, as RNAs lacking the cap are
translated poorly.
• In addition, most eukaryotic mRNAs contain a
short recognition sequence that surrounds the
initiating AUG codon—5'-ACCAUGG Marilyn
Kozak, to function during initiation in the same
way that the Shine-Dalgarno sequence functions
in prokaryotic mRNA. Both greatly facilitate the
initial binding of mRNA to the small subunit of
the ribosome.
• Another difference is that the amino acid
formylmetbionine is not required to
initiate eukaryotic translation. However,
as in prokaryotes, the AUG triplet, which
encodes methionine, is essential to the
formation of the translational complex,
and a unique transfer RNA (tRNAmet ) is
used during initiation.
13.4 Proteins, Heredity, and
Metabolism
• Now let's consider how we know that proteins are
the end products of genetic expression. The first
insight into the role of proteins in genetic processes
was provided by observations made by Sir
Archibald Garrod and William Bateson early in the
twentieth century. Garrod was born into an English
family of medical scientists. His father was a
physician with a strong interest in the chemical
basis of rheumatoid arthritis, and his eldest brother
was a leading zoologist in London. It is not
surprising, then, that as a practicing physician,
Garrod became interested in several human
disorders that seemed to be inherited.
那么基因又是如何实现其功能的？在20
世纪初，英国医生A.Garrod首先发现人
类中几种先天性代谢缺陷疾病，如苯丙
酮尿症（phenylketonuria简称PKU）就
是由一个常染色体隐性基因决定的。
• Although he also studied albinism and cystinuria,
we shall describe his alkaptonuria individuals
afflicted with this disorder cannot metabolize the
aikapton 2,5-dihydroxyphenylacetic acid, also
known as homogentisic acid. As a result, an
important metabolic pathway (Figure 13-10.) is
blocked. Homogentisic acid accumulates in cells
and tissues and is excreted in the urine. The
molecule's oxidation products are black and easily
detectable in the diapers of newborns. The products
tend to accumulate in cartilaginous areas, causing a
darkening of the ears and nose. In joints, this
deposition leads to a benign arthritic condition.
This rare disease is not serious, but it persists
throughout an individual's life.
苯丙氨酸羧化酶
酪氨酸酶
人的先天代谢缺陷
• 1、苯丙氨酸的代谢
蛋白质
蛋白质
苯丙氨酸羧化酶
苯丙氨酸
苯丙酮酸
PKU
PP
苯
丙
尿
症
酮
3,4二羟基
苯丙氨酸
酪氨酸
酪氨酸酶
对羟苯丙酮酸
尿黑酸
尿黑酸氧化酶
AKU
Aa黑尿症
乙酰酯酸
CO2+H2O
cc
白
化
病
黑色素
• 这个代谢途径任何地方出现问题则可能表现：
• （1）黑尿症：病人没有不健康的地方，只是尿液在空气中
变黑。（正常人不会变黑，因为血液中有一种尿黑酸氧化
酶
• （2）白化病：患者不能形成黑色素
• （3）苯丙酮尿症：患者不能形成苯丙氨酸羧化酶——酪氨
酸。由于苯丙氨酸的累积可引起下列变化：
• 过量苯丙氨酸损害中枢神经系统，影响智力发育
• 只能通过苯丙氨酸转氨酶作用——苯丙酮酸，小便排除
• 抑制酪氨酸形成，不能产生黑色素，患者肤色和发色浅。、
• 苯丙氨酸，蛋白质原料，人体必须氨基酸。因此婴儿期及
早诊断明确，可通过食物控制苯丙氨酸的摄入量，防止中
枢神经损害。
• 2、半乳糖代谢
• 不仅氨基酸，脂肪、糖类代谢也可能由于缺乏适当的酶而
受影响。
13.5
The One-Gene:One-Enzyme Hypothesis
• In two separate investigations beginning in 1933,
George Beadle was to provide the first convincing
experimental evidence that genes are directly
responsible for the synthesis of enzymes. The first
investigation, conducted in collaboration with Boris
Ephrussi, involved Drosophila eye pigments.
Together, they confirmed that mutant genes that
alter the eye color of fruit flies can be linked to
biochemical errors that, in all likelihood, involve the
loss of enzyme function.
• Encouraged by these findings, Beadle then joined
with Edward Tatum to investigate nutritional
mutations in the pink bread mold Neurospora crassa.
This one to one
Beadle and Tatum: Neurospora Mutants
• In the early 1940s. Beadle and Tatum chose to work
with Neurospora. By inducing mutations, they
produced strains that had genetic blocks of reactions
essential to the growth of the organism.
• Beadle and Tatum knew that this mold could
manufacture nearly everything necessary for normal
development.
• This organism can synthesize 9 water-soluble vitamins,
20 amino acids, numerous carotenoid pigments, and all
essential purines, and pyrimidines. Beadle and Tatum
irradiated asexual conidia (spores) with X-rays to
increase the frequency of mutations and allowed them
to be grown on "complete" medium containing all the
necessary growth factors (e.g., vitamins and amino
acids, etc.).
• Under such growth conditions,to grow by virtue of
supplements present in the enriched complete
medium. All the cultures were then transferred to
minimal medium.
• If growth on the minimal medium, they able to
synthesize all the necessary growth factors
themselves, and the researchers concluded that the
culture did not contain a mutation.
• If no growth occurred, then they concluded that the
culture contained a nutritional mutation, and the
only task remaining was to determine its type. Both
cases are shown in Figure 13-1 l(a).
• Many thousands of individual spores
derived by this procedure were isolated
and grown on complete medium. To
identify the mutant type, the mutant
strains were tested on a series of different
minimal media [Figure 13-1 l(b) and (c)],
each containing groups of supplements
• Genes and Enzymes:
Analysis of Biochemical Pathways
The one-gene:one-enzyme concept and its attendant
methods have been used over the years to work out many
details of metabolism in Neurospora, Escherichia coli,
and a number of other microorganisms.
• One of the first metabolic pathways to be investigated in
detail was that leading to the synthesis of the amino acid
arginine in Neurospora. By studying seven mutant
strains, each requiring arginine for growth (arg-).
• Adrian Srb and Norman Horowitz ascertained a partial
biochemical pathway that leads to the synthesis of this
molecule. Their work demonstrates how genetic analysis
can be used to establish biochemical information.
• Srb and Horowitz tested each mutant strain's ability to
reestablish growth if either citrulline or ornithine, two
compounds with close chemical similarity to arginine,
was used as a supplement to minimal medium. If either
was able to substitute for arginine, they reasoned that it
must be involved in the biosynthetic pathway of
arginine. They found that both molecules could be
substituted in one or more strains.
Of the seven mutant strains, four of them (arg 4-7)
grew if supplied with either citrulline, ornithine, or
arginine. Two of them (arg 2 and arg 3) grew if
supplied with citrulline or arginine. One strain (arg 1)
would grow only if arginine were supplied; neither
citrulline nor ornithine could substitute for it. From
these experimental observations, the following pathway
and metabolic blocks for each mutation were deduced:
生化突变与一基因一酶说
•
•
•
•
•
•
•
菌株
鸟氨酸
1
—
2-3
—
4-7
生长
基因：
arg1
酶 ：
E1
前体
前体 鸟氨酸
瓜氨酸
精氨酸
—
生长
生长
生长
生长
生长
arg2
arg3
E2
E3
 瓜氨酸  精氨酸。
Insights and Solutions
13.6
One-Gene:One-Polypeptide Chain
• Two factors soon modified the one-gene:one-enzyme
hypothesis.
• First, while nearly all enzymes are proteins, not all
proteins are enzymes. the more accurate
phraseology one-gene :one-protein.
• Second, proteins often show a subunit structure
consisting of two or more polypeptide chains.
Because each distinct polypeptide chain is encoded
by a separate gene, a more modern statement of
Beadle and Tatum's basic hypothesis is one-gene:
one-polypeptide chain.
• These
modifications of
the original
hypothesis
became
apparent
during the
analysis of
hemoglobin
structure in
individuals
afflicted with
sickle-cell
anemia.
• HbA由4条多肽链组成（α2β2），
2α链，各141AA
2β链， 146AA
• Vernon M.Ingram证明HhA和HbS有相同的α链，
只是β链上第6位AA HbA是Glutamine，而HbS是Val。
• 因此HbA和HbS这对等位基因的差别导致了由该基因
所控制的多肽链上的一个氨基酸的差别。
由此可见基因是以某种方式规定了蛋白质中氨基酸顺
序的。
• 已发现由于α链或β链上不同氨基酸的变异导致许
多不同类型的血红蛋白。
• 如HbC也是由于β链上第6位Glu——Lys，纯合的
HbCHbC个体只表现轻度贫血。
• 还有HbHope异常的血红蛋白是由于β链第136位上
氨基酸发生变异，但不产生明显的临床症状。
13.11 Protein Domains and Exon
Shuffling
• We conclude our discussion of proteins by briefly
discussing the important finding that regions made
up of specific ammo acid sequences are associated
with specific functions in protein molecules. Such
sequences, usually between 50 and 300 amino
acids, constitute what are called protein domains
and are represented by modular portions of the
protein that fold into stable, unique conformations
independently of the rest of the molecule.
Different domains impart different functional
capabilities. Some proteins contain only a single
domain, while others contain two or more.
• The significance of domains rests at the tertiary
structure level of proteins. Each such modular unit can
be a mixture of secondary structures, including both a
helicies and p pleated sheets. The unique
conformation that is assumed in a single domain
imparts a specific function to the protein. For example,
a domain may serve as the catalytic basis of an
enzyme, or it may impart the capability to bind to a
specific ligand as part of a membrane or another
molecule. Thus, in the study of proteins, you will hear
of catalytic domains, DNA-binding domains, and so
on. The result is that a protein must be looked at as
being composed of a series of structural and
functional modules. Obviously, the presence of
multiple domains in a single protein increases the
versatility of each molecule and adds to its functional
• An interesting proposal to explain the genetic origin
of protein domains was put forward by Waller
Gilbert in 1977. Gilbert suggested that the functional
regions of genes in higher organisms consist of
collections of exons originally present in ancestral
genes that are brought together through recombination during the course of evolution.
Referring to this process as exon shuffling, Gilbert
proposed that exons, like protein domains, are also
modular. Gilbert proposed that during evolution,
exons may have been reshuffled between genes in
eukaryotes such that different genes share similar
domains.
• Since 1977, a serious research effort has been directed
toward the analysis of gene structure. In 1985, more
direct evidence in favor of Gilbert's proposal of exon
modules was presented. For example, the human gene
encoding the membrane receptor for low-density
lipoproteins (LDL) was isolated and sequenced. The
LDL receptor protein is essential to the transport of
plasma cholesterol into the cell. It mediates endocytosis
and is expected to have numerous functional domains.
These include the capability of this protein to bind
specifically to the LDL substrates and to interact with
other proteins at different levels of the membrane
during transport across it. hi addition, this receptor
molecule is modified post-translationally by the
addition of a carbohydrate; a domain must exist that
links to this carbohydrate.
Detailed analysis of the gene encoding this protein supports the
concept of exon modules and their shuffling during evolution,
The gene is quite large—45,000 nucleotides—and contains
18 exons. These represent only slightly less than 2600
nucleotides. These exons are related to the functional
domains of the protein and appear to have been
recruited from other genes during evolution.
Figure 13-21 shows these relationships.
• The first exon encodes a signal sequence that is
removed from the protein before the LDL receptor
becomes part of the membrane. The next five exons
represent the domain specifying the binding site for
cholesterol. This domain is made up of a sequence of
40 amino acids repeated seven times. The next domain
consists of a sequence of 400 amino acids bearing a
striking homology to the mouse peptide hormone
epidermal growth factor (EOF). This region is encoded
by eight exons and contains three repetitive sequences
of 40 amino acids. A similar sequence is also found in
three blood-clotting proteins. The fifteenth exon
specifies the domain for the posttranslational addition
of the carbohydrate, while the remaining two specify
regions of the protein that are part of the membrane,
anchoring the receptor to specific sites called coated
pits on the cell surface.