Download Protein translation - San Diego Mesa College

SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Protein translation

the last step of the conversion of the genetic information (stored in a linear
nucleotide sequence) on the DNA and transcribed into a single-stranded
messenger molecule (mRNA) into a functional polypeptide chain (called
protein or enzyme) biologists call protein translation
mRNA

polypeptide chain

the process of protein translation occurs at special protein structures in the cell,
called ribosomes
 ribosomes are the cell’s polypeptide manufacturing belt

in comparison to cell organelles, ribosomes are small, but not less complex
structures, consisting of two unequally sized sub-units, referred to as large and
small sub-units, which fit closely together as seen in the Figures below

each ribosomal sub-unit is composed of a complex between RNA molecules and
proteins; each sub-unit contains at least one ribosomal RNA (rRNA) sub-unit and
a large quantity of ribosomal proteins

ribosomes in bacteria are located in the cytosol, while in eukaryotic cells they
are found on the surface of a special organelle, the rough endoplasmic
reticulum (rER)
The location of ribosomes in a eukaryotic cell
1
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.

ribosomes consist of 2 major components:
1.
Protein
 the protein part consists of two globular protein sub-units, a so-called
small and large sub-unit
 the protein sub-units have a different size in bacteria and eukaryotic cells
(see Figures below)
2.
rRNA
 three rRNA molecules (5S, 16S & 23S-rRNA) ( in prokaryotes) or
 four rRNA molecules (7S, 18S & 28S-rRNA) ( in eukaryotes),
each with a different are attached with certain regions of both
protein sub-units

more than half of the weight of a ribosome is RNA, and there is clear evidence
that the ribosomal RNA (rRNA) molecules have catalytic activity; rRNA plays a
central part in the attachment of amino acids and the extension of the
polypeptide chain
The ribosome structures
 the Figures show only the protein sub-units of a eukaryotic (top)
and a bacterial (bottom) ribosome
2
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.

during the cellular process of translation, several components or molecules dock
at these two ribosomal structures

a ribosome contains three binding sites for RNA molecules:
- one for mRNA and two for tRNAs
- one site, called the peptidyl-tRNA-binding site, or P-site, holds the tRNA
molecule that is linked to the growing end of the polypeptide chain
- another site, called the aminoacyl-tRNA-binding site, or A-site, holds the
incoming tRNA molecule charged with a covalently bound amino acid (see
Figure below)
I.
-
2 amino acid-loaded tRNA (= aminoacyl tRNA) molecules
they bind to the so-called P and A sites of the small subunit
the P site holds the growing polypeptide chain
the A site is the docking site for the new and matching tRNA molecule with its
attached amino acid (= aminoacyl-tRNA) bacterial ribosome during protein
synthesis
II. one mRNA strand
- binds also to the small sub-unit of the ribosome
- its correct alignment with the A- and P-sites is enabled with the help of the
ribosomal RNA (= rRNA)
- the 2 anti-codon sequences of two closely neighbored and ‘loaded’ tRNA
molecules base-pair with the corresponding codon on the bound mRNA strand
- all three biomolecules are closely brought and hold together in a cavity build by
the two ribosomal sub-units
- the ribosome assures that the 2 anti-codon sequences of two closely neighbored
and amino acid-‘loaded’ tRNA molecules base-pair with the corresponding codon
on the bound mRNA strand
 all three biomolecules are closely brought and hold together in a cavity
build by the two ribosomal subunits
 at the begin of the translation process, the mRNA binds to the small ribosomal
sub-unit via a so-called recognition sequence located at the begin and end of
the RNA strand
The tRNA- & mRNA-binding sites on the ribosome & RNA alignment
aa = amino acid
3
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
3 -dimensional prokaryotic ribosome structure
 computer-assisted image based on X-ray crystallographic data with a purified
ribosome
Large ribosomal
sub-unit
Small ribosomal
sub-unit
tRNA (waiting)
tRNA (A-site)
tRNA (P-site)
The 3 major steps of translation of mRNA at the ribosome

generally, translation proceeds in a highly ordered process. First all components
necessary for translation are brought together in a process called Initiation.

Secondly, in a process called Elongation, the polypeptide chain grows in a
distinct direction and becomes longer.

Finally, the chain of amino acids gets released from the ribosome in a process
called Termination
1. Initiation or codon recognition
- a so-called pre-initiation ribosome complex forms; it consists of:
1.
2.
3.
4.
-
the small ribosome sub-unit (= 40S)
the mRNA
a GTP molecule
an initiation protein/factor (= eIF-2)
the large ribosomal sub-unit (= 60S) associates with this pre-initiation
complex to form the 80S complex
this step requires another protein factor, called eIF-5
4
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
-
in prokaryotes, the 16S-rRNA component of the ribosome recognizes the socalled Shine-Dalgarno sequence on the “captured” mRNA strand
the anti-codon of a “loaded” or so-called amino-acyl tRNA molecule basepairs with its ‘correct’ mRNA codon in the A-site of the ribosome
this first step requires in both, Prokaryotes and Eukaryotes, a specific
initiator-tRNA, called tRNAmetI
 in prokaryotes the methionine of the initiator-tRNA is formylated
Initiator tRNAs:
Bacteria and eukaryotic cells contain two different methionine tRNAs:
1. tRNAiMet
 initiates translation at first AUG of gene
 binds on P-site of small ribosomal subunit
 in bacteria, methionine of Met-tRNAiMet is formylated
2. tRNAMet
 incorporates methionine only into a growing
protein chain
binds on ribosomal A-site
-
-
the initiator tRNA molecule base-pairs with the AUG start codon of the
mRNA strand in the P-site of the ribosome; it incorporates the initial amino
acid methionine into the new protein
Initiation requires so-called Initiation factors (IF); IF’s are proteins which
enable the correct and accurate positioning of certain players at the ribosome
 e.g. eIF-1, eIF-2, eIF-4E (or GEF), eIF-4E, eIF-4A
2. Elongation or peptide bond formation
-
-
-
-
during elongation the new polypeptide strand forms and becomes longer (=
elongates)
Elongation at the ribosome occurs in a cyclical manner by step-wise addition
of new aminoacyl- tRNAs in the A- site of the assembled bacterial 70S or
eukaryotic 80S ribosome – Met-tRNAiMet complexes
Elongation requires the presence of special proteins called elongation factors
(EFs)
at the end of one complete round of amino acid addition, the A-site of the
ribosome will be empty again and the ribosome ready for the docking of the
new, matching amino acyl-tRNA molecule
the matching aminoacyl-tRNA (= the aminoacyl-tRNA with the matching anticodon site) is brought to the empty A-site of the ribosome by an elongation
factor (EF) complex, called eEF-1-GTP
the amino acid/peptide attached with the tRNA in the P-site is transferred to
the amino group of the aminoacyl-tRNA located in the A-site; this step of
translation, which is also called transpeptidylation, is catalyzed by an
enzyme, called peptidyl transferase
 at least parts of this chemical reaction seem to be catalyzed by the
enzyme-activity of the 23S-rRNA !! (RNA can work as an enzyme =
5
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
-
ribozyme activity !!)
the polypeptide strand separates from the tRNA molecule in the P-site and
attaches to the amino acid bound on the other tRNA in the A-site
a covalent peptide bond is formed between the two neighboring amino acids
Translocation
- the now ‘empty’ tRNA in the P-site leaves the ribosome and the ribosome
moves the tRNA (including the attached polypeptide chain) in the A-site
over to the vacant P-site
- this step is again catalyzed by an enzyme, called eEF-2 and requires energy
in form of the ATP-like molecule GTP
 during this translocation the anti-codon of the remaining tRNA remains paired with
its codon on the mRNA
-
-
since the mRNA also moves over to the P-site it exposes the next codon to
the anti-codon of the new incoming and ‘loaded’ tRNA
the process of polypeptide elongation starts again with step 1 until a stop
codon on the mRNA strand arrives at the ribosomal A-site
following this translation scheme, the whole mRNA molecule is translated in
5’  3’ direction!
the different steps of elongation in a nutshell:
1. Second aminoacyl-tRNA bound to EF1 α-GTP (eukaryotes) or EF-Tu –
GTP ( bacteria) binds to the vacant A-site of the ribosome
2. Peptide bond formation occurs between the α amino
group of 2. amino acid and the “activated” (aminoacylated) methionine
attached to the initiator tRNA  “Transpeptidylation reaction”
 catalyzed by 23S rRNA of large ribosomal subunit
3. the ribosome is moved one codon distance (= 3 nucleotides long) along
the mRNA (“Translocation”); the second amino acyl tRNA in A-site moves
over to the P-site
 this step of protein translation requires EF2-GTP (eukaryotes) or
EF-G-GTP (bacteria)
 GTP hydrolysis supplies energy for translocation
4. The now empty A- site is “re-loaded” with the next aminoacyl t-RNA … etc.
6
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
The elongation cycle of cellular translation
3. Termination
-
-
-
the signals for ending translation at the ribosome are the same for
Prokaryotes and Eukaryotes
in both cases, the termination signal is set by the appearance of a
combination of the so-called stop codons at the 3’-end of the RNA molecule;
the stop codons are UAG, UUA and UGA
the termination codons are recognized by certain so-called releasing factors
(= eRFs)
the releasing factors (eRF1 or eRF-2) bind to the empty A-site on the
ribosome; as a consequence the peptidyltransferase moves the peptide chain
(bound in the P-site) to water instead of an aminoacyl-tRNA!
the complete polypeptide chain (approx. 100 amino acids long!) leaves the
ribosome which is freed from the last tRNA molecule in the P-site
the inactive ribosome releases its bound mRNA and separates into its two
sub-units again
7
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Termination… the final scenario of protein translation

Since the ribosomes of eukaryotic cells are located on the membrane of the rER,
the newly formed polypeptide chain doesn’t leave into the cytosol but is tunneled
through the phospholipid membrane of the rER into the interior space (= lumen)
of the rER
- in the rER lumen, the freshly synthesized polypeptide chain is properly folded
into its final functional 3-dimensional shape with the help of special folding
helper enzymes, called chaperones
- most of the polypeptides or proteins are even further trimmed (= processed)
within the rER lumen in a cellular process called post-translational
modification
e.g. in many cases sugar molecules are attached to the proteins which is
called glycosylation; protein glycosylation plays a fundamental role in
the friend or foe-recognition process of our immune system or in the
formation of the human ABO blood groups
e.g. many so-called signaling proteins become modified by attachment of
certain fatty acids in a process called farnesylation or palmitoylation
e.g. some polypeptides, such as insulin, are processed by chopping parts
of the chain off before they are released into the cell or blood stream
 the ‘cellular end control’ for newly synthesized proteins is also found in the
interior space of the rER;
-
defect or not properly folded proteins are recognized and sorted-out by
‘cellular watchdogs’ called heat shock protein (hsp);
they are then send to another organelle, called proteasome, where they are
degraded and recycled
8
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.

Usually mRNA molecules are translated simultaneously by a number of
ribosomes on the rER
Ribosomal translation is a tremendously fast cellular process
it takes a cell less than a minute to synthesize an average-sized polypeptide of
about 100 amino acids!

DNA transcription and translation is usually not permanently happening in a cell
and does not involve all cellular genes at the same time

Both processes are rather highly regulated cellular processes which are
controlled at different levels
Regulation of protein translation

cells can control the expression of genes and it’s products also on the level of
protein translation at the ribosomes

this is primarily achieved through tight control of the activity of protein factors of
the ribosomal “protein translation machinery” by following three mechanisms
1. Control by protein phosphorylation of initiation factors
- phosphorylation of eIF-2 by the heme-controlled inhibitor (HCI) blocks the
essential exchange of the bound GDP for GTP
2. Control by Interferons (IFs)
- interferons are cellular signaling molecules that activate RNA-dependent
protein kinases (= PKRs)
- the IF-activated PKRs phosphorylate and inactivate eIF-2 therefore
preventing initiation of protein translation
- most IFs are released from white blood cells after viral attack
- production and release of IFs is induced by many viruses and dsRNA
molecules
3 major classes of interferons are known
-Interferon produced in leukocytes
-Interferon  produced in fibroblasts
-Interferon  produced in lymphocytes
3. Control after protein translation
- most prominently through post-translational protein modification, such as
1. Glycosylation (= attachment of sugar residues to proteins)
2. Isoprenylation/Acylation (= attachment of isoprenoid or fatty acid
Residue to protein)
3. Ubiquitination & Proteolytic Digest
(Proteasome, Apoptosis  Caspases)
9
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Protein translation & Mistakes or Errors

As seemingly perfect the cellular “protein translation machinery” may appear to
the human observer, it is not free of eventual flaws and errors occurring during
the intricate protein translation process at the ribosome

Mistakes in translation of the genetic information of the mRNA molecule into a
corresponding amino acid sequence occurs most frequently at the third codon
position

The third codon position (e.g. the G in CUG) is the most error prone spot of the
codon since it has the weakest binding affinity for the corresponding tRNA
molecule
“The third codons form the weakest link of the genetic code…”

Scientists say, that the genetic code “wobbles” in the third codon position

Since, interestingly, codons sharing 2 out of 3 bases tend to code for amino acids
with the same physico-chemical properties (e.g. hydrophobicity, polarity, net
charges), the consequences of a “codon wobbling event” are usually minor for
the resulting amino acid chain

The surprising non-random codon arrangement of the genetic code (see sections
above) assures that the consequences of a wobbling event in the third codon site
during protein translation does NOT lead to an incorporation of an amino acid
with completely different physico-chemical properties

Based on this interesting observation, whereas the genetic code of life bears a
non-random assignment of codons with their corresponding amino acids,
scientists hypothesize today, that the genetic code is the product of natural
selection on the molecular level

The genetic code is dynamic and evolved through natural selection

Of all possible random assignments of 64 codons with the existing 20 amino
acids, the genetic code unraveled by M. Nirenberg, turned out to be the very
code used by almost all biological organisms on planet Earth to assure the least
risk of producing aberrant and dysfunctional proteins during protein translation
due to intrinsic “wobbling”, mutations or viruses

Today, we have to assume that not only the intricate shapes, morphologies and
traits of biological organisms evolved over long periods of time, but that even the
genetic code has to be seen as the product of evolution

The genetic code evolved by natural selection as the most favorable of all
possible genetic codes to life and survived the billions of years since the rise of
the first life forms on planet Earth
10
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Further Readings:
1. H. Lodish, A. Berk, S.L. Zipursky, P. Matsudaira, D. Baltimore & J. Darnell.
“Molecular Cell Biology” (4th edition: W.H. Freeman): Chapter 4.
2. S.J. Freeland & L.D. Hurst “Evolution Encoded” Scientific American (April
2004): 84-91
3. S.J. Freeland & L.D. Hurst. “The genetic code in one in a million.” J.
Molecular Evolution 47(3): 238-248 (1998)
Inhibition of protein translation by antibiotics and toxins

Since DNA transcription and protein translation are enormously complex cellular
process, involving many “molecular players”, it is not too surprising that both
processes are enormously vulnerable to many natural or synthetic compounds
 many of these transcription or translation blockers (or inhibitors) we
humans know and fear (some since ancient times) as famous or infamous
toxins or poisons

many of our modern antibiotics, we use for treatment of bacterial or fungal
infections, function through blockade (= inhibition) of cellular translation

different antibiotics interfere and block at different levels of translation ( see
Table below)
11
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Known Inhibitors of Cellular Protein Translation
Antibiotic or Toxin
C
Chhlloorraam
mpphheenniiccooll
S
Sttrreeppttoom
myycciinn
TTeettrraaccyycclliinnee
E
Erryytthhrroom
myycciinn
Target of protein translation

inhibits the bacterial peptidyl
transferase

inhibits the prokaryotic peptide chain
initiation

inhibits the binding of the
aminoacyl-tRNA to the ribosome
small unit in prokaryotes
inhibits the translocation of the
mRNA through the ribosome large
sub-unit in prokaryotes
resembles in shape an aminoacyltRNA molecule and interferes with
peptide transfer; leads to premature
termination at the ribosome
attaches an ADP-ribose molecule to
the eEF-2 in eukaryotic cells and
inactivates this elongation factor
this plant-derived toxin catalyzes the
cleavage of the eukaryotic large subunit rRNA from the ribosome
inhibits the eukaryotic
Peptidyltransferase


P
Puurroom
myycciinn
D
Diipphhtteerriiaa ttooxxiinn
R
Riicciinn
C
Cyycclloohheexxiim
miiddee



The genetic code of life

not like in computers, where technical engineers use a 2 numbered, so-called
binary code, to encrypt and save technical data and information, all biological
organisms use a sequence of 3 nucleotides on the DNA/RNA molecule - or a socalled triplet - code to encode the information of life
- each triplet code stands for an individual amino acid
- the 3 nucleotide unit is also called a codon
e.g.
GCT
A G G
GTC

TTrriipplleett ccooddoonn
aam
miinnoo aacciidd 11
aam
miinnoo aacciidd 22
aam
miinnoo aacciidd 33 . . . etc.
12
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.

the whole genetic information of an organism is written down as (triplet) codons,
which are translated (= translation!) into an amino acid sequence
- the sequence of triplet codons is the “chemical language” of DNA and RNA

triplets (not duplets) of bases provide the smallest unit of information which is
able to specify all the known 20 amino acids
- duplets could only code for 42 = 16 different amino acids, while a triplet code
enables 43 = 64 different code words for amino acids!

since the triplet code gives the cell more coding possibilities than it actually
needs to code, there are multiple triplets coding for the same of the existing 20
amino acids; scientists say the genetic code is redundant

the different triplets coding for a specific amino acid are written down in the socalled genetic code of the DNA molecule
Milestone experiments of science
 The genetic code was deciphered by the elegant and milestone experiments
conducted by the American biochemist M. Nirenberg in 1961
- he chemically synthesized short, artificial RNA molecules with defined
sequences (e.g. poly-U or poly-UUC) and used these in a (by that time
established) cell-free transcription system
- a cell-free transcription system consists of ribosomes and essential
biomolecules ( co-factors)

by varying the RNA sequences and determining the incorporated amino acids of
the resulting polypeptide chain, Nirenberg retrieved the 61 of the 64 theoretically
possible triplet codes for each amino acid; he unraveled the genetic code
(see Chart below)
 he revealed that the codon A
AU
UG
G has dual function:
1.
2.
it codes for the amino acid methionine
it is the start signal or ssttaarrtt codon for the transcription machinery

today we know, that ALL polypeptides manufactured in living organisms, start
with the amino acid methionine!

UA
the three other codons (U
AA
A,, U
UA
AG
G and U
UG
GA
A) are not coding for an amino acid;
they set the stop signal for the transcription enzymes; they are also called ssttoopp
codons

almost all of the genetic code is shared by all organisms on Earth; from the
simplest bacteria to the most complex plants and animals; different forms of life
use the same genetic code to translate their DNA sequence into a protein
- that means that bacteria or yeast can translate human genetic messages into
functional proteins
- this is of great importance for modern Biotechnology, where bacteria or yeast
cells are used to produce large amounts of proteins or peptides for e.g. bioanalytical tests or treatment of human disease, e.g.:
13
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Erythropoietin (EPO)
Interferons
Insulin



anemia
certain cancers
diabetes
The genetic code & Evolution
“The genetic code is not frozen in time but obviously evolves …”

in the recent years, however, scientists identified at least 16 variants to the code

while in most biological organisms the codon CUG is translated into the amino
acid leucine (Leu), several biological species have been discovered where this
codon translates into a different amino acid
- e.g. in the fungal Candida species, CUG translates into serine (Ser)
- e.g. the green algae Acetabularia translates the stop codons UAG & UAA into
glycine (Gly)
- e.g. in some organisms the standard stop codon UGA codes for the rare
amino acid selenocysteine
- e.g. mitochondria and many bacterial species have large variations to the
“classical genetic code” unraveled by Nirenberg
 in the yeast mitochondrial code, 4 of 6 codons that normally encode
leucine (Leu) code for threonine (Thr)

today it becomes more and more obvious, that the genetic code is not a “static
product of chance” with randomly assigned codons for each of the 20 amino
acids, but rather the product of a molecular evolutionary process

the assigned codons are arranged well in terms of ensuring that errors occurring
during protein translation do not lead to catastrophic consequences, means the
synthesis of dysfunctional proteins
The genetic code chart
14
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.

with the knowledge of the complete DNA sequences of the genomes of many
organisms, including humans, we are now able to read the genetic information
and master plans for all life components of these species, a knowledge which
may one day pave the way for the cure of heritable genetic diseases, such as
cystic fibrosis, muscular dystrophy, SCID, and many others, in humans or the
development of plants with improved traits with the help of gene therapy
15