Download Chapter 10: DNA transcription, protein synthesis

SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
Chapter 10: DNA transcription, protein synthesis &
The genetic code
- Part II -
“ DNA transcription and ribosomal protein translation are the crucial cellular
processes which guarantee the controlled flow of genetic information from the
digital molecular code of DNA to the final cellular protein product.”

the information for the shape and function of the cell’s and organism’s proteins and
enzymes is laid down in a molecular (or genetic) code contained in the DNA
molecule in form of coded units called genes

the scientific study of the molecular gene reading and genetic code-translating
process occurring in cells of all biological organisms on planet Earth lead to the
discovery of two of the most fascinating and elementary biological processes – DNA
transcription and protein translation

our modern understanding of these two biological processes came a long and
difficult way in human history
H
Hiissttoorryy

1909: the English physician A. Garrod suggests from his observations with patients
suffering from certain metabolic diseases that genes dictate the phenotype (= the
health condition) of a person
 11994400:: the American geneticists G. Beadle and E. Tatum formulate the one gene 
one enzyme hypothesis based on their experimental results with so-called
nutritional mutants of the bread mold Neurospora crassa
 each of the nutritional mutants lacked one specific enzyme and could only grow
on a medium which was supplemented with that nutrient

later this hypothesis proofed to be correct and was extended on proteins as well

in 1961, the American biochemist M. Nirenberg and co-worker decipher the genetic
code which is shared by all forms of life on planet Earth; for this scientific mile stone
work he received the Nobel prize in Medicine
 he chemically synthesized short, artificial RNA molecules with defined sequences
(e.g. poly-U or poly-UUC) and used these in a cell-free transcription system
consisting of ribosomes and essential factors;
 ttooddaayy we know that the DNA molecule is organized into special functional units, the
so-called genes and each gene on the DNA double helix bears the information for
one specific protein or enzyme
1
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
Introduction:

the genetic information - laid down in the long sequence of bases, e.g.
AGCCGTTAACGT, within the DNA molecule - encodes for a polypeptide chain
 protein or enzyme can consist of two or more polypeptide chains
 e.g. the insulin receptor (= a protein which is responsible for the regulation of
glucose uptake in our body) is build of two different polypeptide chain which are
attached with each other

to become a cellular protein or enzyme, this genetic code laid down in the nucleotide
sequence of DNA molecules has to be somehow translated by the cell

as a pre-requisite of this so-called translation of the genetic information of a gene
into a protein, a cell first copies the DNA into a single-stranded polynucleotide
molecule, called RNA

this special form of DNA replication is called DNA transcription
Definition: DNA transcription
- transcription is the cellular writing of the genetic information of a gene on the DNA
double helix into a piece of RNA molecule

let’s look at this first step on the way from genes to cellular proteins in more detail
DNA Transcription

Transcription = the replication of the genetic information of a gene on the DNA
strand into an RNA molecule
DNA

RNA

DNA transcription occurs in the cell nucleus of eukaryotic cells

Specific segments of bases along the DNA strand, each with a defined beginning
and an end, mark a so-called gene

Each gene harbors the genetic information of the DNA strand and codes for a
specific protein or enzyme

the complete genetic information of an organism is called the genome, which
consists of many genes
- e.g. the genome of wild mustard (Arabidopsis thaliana) codes for approx.
25,000 genes and contains about 130 million base pairs
- e.g. the human genome has an estimated number of 50,000 – 100,000 genes
(!) consisting of approx. 3 billion (!!) nucleotides
 despite this huge number, the human genome has recently been announced
to be completely decoded (= sequenced) by a joined effort of American and
European research teams!
2
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
TThhee w
waayy ffrroom
m aa ggeennee ttoo aa cceelllluullaarr pprrootteeiinn

during DNA transcription, only one strand of a the double-helix of a gene, the socalled sense-strand, serves as the so-called template for DNA transcription to form
the new RNA molecule
- certain enzymes unwind and open the DNA double helix and distinct places and
create single stranded DNA regions
- the distinct places along the DNA strand where transcription begins are called
transcription start sites

along these unwind and single-stranded DNA regions, new nucleotides are paired
according to the Watson-Crick base-pairing rule ( A with T; and G with C)

the polymerization of the new nucleotides during the process of DNA transcription is
catalyzed by an enzyme called RNA polymerase

the RNA polymerase recognizes certain structures on the DNA strand, called
enhancers and promoter regions, which tells it where to dock on and where to
start the transcription process
- In order to begin transcription, RNA polymerase requires a number of so-called
transcription factors (TFs), e.g. TFIIA, TFIIB, etc.
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SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
-
the RNA polymerase plus the transcription factors recognize and bind to the socalled TATA box

these enhancer and promoter regions are always located downstream of the of the
so-called transcription start site of a gene
- note: the transcription start site of each gene lays in front of a typical ATG triplet
nucleotide sequence (= the so-called start codon) which dictates the begin of the
later polypeptide chain to be synthesized
- the promoter region also dictates which of the two DNA strands is going to be the
sense strand

after docking to the DNA strand, the RNA polymerase reads only the so-called
sense strand of the double-stranded DNA molecule
- this is divergent to the earlier introduced features of the DNA- polymerase, which
reads both strands of the DNA molecule
- an additional divergence to the DNA polymerase is, that the RNA polymerase
base-pairs an uracil (U) instead of a thymine (T) with adenine (A) along the DNA
strand

the resulting RNA molecule is (as the daughter DNA strands after DNA replication)
an exact copy of the DNA sense strand sequence

The sequence of nucleotides along the RNA molecule dictates the later sequence of
amino acids in the polypeptide chain ( see protein translation in the following
section)
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SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
The first events of cellular transcription
Promoter region
ttrraannssccrriippttiioonn
ssttaarrtt ssiittee

gene

docking of first transcription
factor (TFII)

docking of second
transcription factor (TFII)

docking of R
RN
NA
A ppoollyym
meerraassee
+ other factors

ATP-dependent
phosphorylation of
R
RN
NA
A ppoollyym
meerraassee
5
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
TThhee 33 sstteeppss ooff D
DN
NA
A ttrraannssccrriippttiioonn

after activation of the RNA polymerase II by attaching phosphate groups (=
phosphorylation), DNA transcription proceeds in three major steps

The 3 steps of DNA transcription
11.. IInniittiiaattiioonn

An activated RNA polymerase recognizes and binds to a unique DNA sequence
located just in front of a gene called the promoter region;

It unwinds the helix of the DNA molecule in that region and starts adding new
nucleotides along the single-stranded ‘sense- DNA strand’; following the WatsonCrick base-pairing rule

The RNA polymerase separates the DNA strands at the appropriate point and
bonds the RNA nucleotides as they base-pair along the DNA template.
- Like DNA polymerases, RNA polymerases can add nucleotides only to the
3’ end of the growing polymer;
- genes are transcribed in 3’->5’ direction, creating a 5’->3’ RNA molecule

Bacteria have a single type of RNA polymerase that synthesizes all RNA
molecules. In contrast, eukaryotes have three RNA polymerases (I, II, and III) in
their nuclei. In Eukaryotes, RNA polymerase II is used for mRNA synthesis.

Initiation is a crucial step during DNA transcription since it involves many
regulatory proteins, most importantly the highly regulated transcription factors
(TFs) (see Figure above & ‘regulation of gene expression’ for more details)
22.. E
Elloonnggaattiioonn

the RNA synthesis is fully running

As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20
bases at time. The enzyme adds nucleotides to the 3’ end of the growing singlestranded mRNA strand.

Behind the point of RNA synthesis, the double helix re-forms and the RNA
molecule peels away.

the newly formed RNA molecule peels-off the single-stranded DNA template
- the two single DNA strands join together again and form the double helix
6
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
33.. TTeerrm
miinnaattiioonn

The RNA polymerase proceeds with DNA transcription until it reaches a
sequence of bases, the so-called terminator sequence, which consists of a
series of stop codons (= UAA, UGA and UAG)

In prokaryotes (bacteria), RNA polymerase stops transcription right at the end of
the terminator. Both the RNA and DNA is then released.

In eukaryotes, the polymerase continues for hundreds of nucleotides past the
terminator sequence, AAUAAA.

At a point about 10 to 35 nucleotides past this sequence, the pre-mRNA is cut
from the enzyme.

The RNA polymerase detaches from the newly formed RNA molecule and the
corresponding gene
7
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
The three steps of DNA transcription

3 different kinds of RNA molecules are assembled (= polymerized) by this
transcription mechanism
1.
messenger RNA (= mRNA)
2.
transfer RNA (= tRNA)
3.
ribosomal RNA (= rRNA)
8
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.

all three play a crucial role in the final cellular scenario on the way from the gene
to a functional protein or enzyme
1. TThhee m
meesssseennggeerr R
RN
NA
A ((== m
mR
RN
NA
A))

mRNA is a single-stranded polynucleotide strand, which delivers the encoded
amino acid sequence of the future protein or enzyme out of the nucleus to the
ribosomes
 in eukaryotic cells, the mRNA leaves the cell nucleus via the nuclear openings
or pores

in eeuukkaarryyoottiicc cceellllss, however, only a shortened version or a so-called spliced
form of the original RNA strand (= nRNA or pre-mRNA) reaches the ribosome as
mRNA

during RNA splicing, the so-called Intron sequences are cleaved or cut out of
the primary DNA transcription product (= pre-mRNA); only the assembled exon
sequences are send to the ribosomes

in the cell nucleus of eukaryotic cells, only about 1% (!!) of the DNA actually
encodes for a certain protein or enzyme product; these coding sequence of
cellular DNA are called exons, they are interrupted by long stretches of noncoding DNA, the so-called introns

during DNA transcription into RNA, the cell first transcribes all of the DNA,
including the exons and introns of the gene, into a complementary RNA copy;
this primary transcription product is called ‘nuclear RNA’ (= nRNA) or pre-promessenger-RNA

the cell then removes in a second step all information-less (= non-coding) introns
from the mRNA by a process called RNA splicing; this edited ( or spliced RNA)
sequence is called pro-mRNA

the spliced mRNA (or so-called pro-mRNA) molecule is further processed and
trimmed by the cell to prevent rapid degradation of the molecule by cellular
enzymes called RNAses

At the 5’ end of the pre-mRNA molecule, a modified form of guanine is added, to
form the socalled 5’ cap. This chemical modification helps to protect mRNA from
hydrolytic enzymes. It also functions as an “attach here” signal for ribosomes.

Other enzymes attach consecutive adenine nucleotides to the 3’-end of the promRNA molecule to form a so-called poly-A sequence or poly-A tail in a process
called poly-adenylation
- both modified mRNA end modifications help to prevent the otherwise very
rapid degradation of this very ‘fragile’ cellular molecule
9
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.

The thus spliced and processed final version of the mRNA travels from the
nucleus into the cytosol to the ribosomes; the place of cellular protein synthesis,
called ribosomes
- in eukaryotic cells the ribosomes are located on the surface of the rough
endoplasmic reticulum (rER)
- in bacteria they swim freely in the cytosol
The Intron-Exon structure of eukaryotic DNA
10
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
11
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
Splicing of eukaryotic, but not prokaryotic RNA
S
Scchheem
mee:: E
Euukkaarryyoottiicc R
RN
NA
AS
Spplliicciinngg
12
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
2. The transfer-RNA (= tRNA)

the tRNA molecule is a short polynucleotide chain consisting of only about 80
nucleotides

the tRNA molecules function as the cellular interpreter and decoder molecules
which translate the genetic language (= genetic code) into the amino acid
language (= sequence)

‘t’ stands for transfer, because tRNAs transport and transfer the cellular amino
acids to the mRNA strand, which has docked at the ribosomes

at the ribosomes they convert the 3 letter codes (= codon) of the nucleotides on
the mRNA molecule into the one-letter amino acid words

a tRNA molecule picks up “its amino acid’ and transfers it to its corresponding
codon on the mRNA strand (following the genetic code)

tRNAs have a complex 3-dimensional structure which has two major so-called
interaction domains (see Figure below)
11..
an amino acid attachment site
 the site on the tRNA molecule where the corresponding amino acid
is covalently attached to the tRNA
2.
a so-called anti-codon site
 which is complementary to a codon triplet on the mRNA strand with
which it base-pairs during the translation process

the transfer of the matching amino acid onto the corresponding tRNA is
preformed by a specialized enzyme under consumption of ATP
- this tRNA-amino acid complex then delivers its load to the growing
polypeptide chain at the ribosome
13
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
Functional domains (left figure) &
3-dimensional structure (right figure) of a tRNA molecule
aam
miinnoo
aacciidd-attachment site

m
mR
RN
NA
A
_______ GCU_____________
(codon)

aannttii--ccooddoonn
site
3. The ribosomal RNA molecule (= rRNA)

it is the RNA molecules which are crucial structural and functional part of the
ribosomes; it actively takes part in the many catalytic activities of this proteinsynthesizing protein complex
- two forms of rRNA with different length exist in prokaryotic organisms (= 16SrRNA & 23S-rRNA) and in eukaryotic organisms (= 18S-rRNA & 28S-rRNA)
- they are synthesized (= transcribed) in high amounts in a special region of the
nucleus, called the nucleolus
14
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
Micro-RNA
-
-
in the past years, scientist discovered additional forms of RNA molecules in
cells of fungi, plants and animals, the small, so-called micro-RNAs, which
mostly unknown biological function
the so-called silencer RNA or (siRNA) molecules seem to have an important
function in the shutting-off (= silencing) of gene expression and to prevent
viral replication in cells
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.

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)
15
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.

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.:
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)
16
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.

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

with the knowledge of the complete DNA sequences for many organisms,
including humans, we are now able to read the genetic information and master
plans for all life components of these species
17
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
P
Prrootteeiinn ttrraannssllaattiioonn –– TThhee eevveennttss aatt tthhee rriibboossoom
mee

the last step of the conversion of the genetic information (stored in a linear
nucleotide sequence) on the DNA into a functional polypeptide chain (called
protein or enzyme) biologists call protein translation

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

a 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
18
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); 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

many more proteins are known which interact or bind to the ribosome
The ribosome structures
 the Figures show only the protein sub-units of a eukaryotic (top)
and a bacterial (bottom) ribosome
19
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); 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 three 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)
- the empty (E) site holds the discharged tRNA which gave off its bound amino
acid during the transpeptidylation process
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
20
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
-
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
21
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); 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)
22
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
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. IInniittiiaattiioonn 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
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)
-
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
23
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
Translation Initiation
22.. E
Elloonnggaattiioonn or peptide bond formation
-
-
-
-
during elongation the new polypeptide strand forms and becomes longer (=
elongates)
Elongation at the ribosome occurs in a cyclical manner
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 =
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
24
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
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!
25
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
The elongation cycle of cellular translation
26
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
27
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
The three steps of elongation
33.. TTeerrm
miinnaattiioonn
-
-
-
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
28
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
-
the inactive ribosome releases its bound mRNA and separates into its two
sub-units again
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);
29
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.

they are then send to another organelle, called proteasome, where they are
degraded and recycled
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
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

A point mutation of DNA that results in replacement of a pair of complimentary
nucleotides with another nucleotide pair is called a base-pair substitution.
Some base-pair substitutions have little or no impact on protein function.
30
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.

In silent mutations, alterations of nucleotides still indicate the same amino acids
because of redundancy in the genetic code.
- Other changes lead to switches from one amino acid to another with similar
properties
- Still other mutations may occur in a region where the exact amino acid
sequence is not essential for function

Other base-pair substitutions cause a readily detectable change in a protein.
These are usually detrimental but can occasionally lead to an improved protein or
one with novel capabilities. Changes in amino acids at crucial sites, especially
active sites, are likely to impact function.
31
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.

Missense mutations are those that still code for an amino acid but change the
indicated amino acid.

Nonsense mutations change an amino acid codon into a stop codon, nearly
always leading to a nonfunctional protein.
32
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.

Insertions and deletions are additions or losses of nucleotide pairs in a gene.
These have a disastrous effect on the resulting protein more often than
substitutions do.
- many inherited human disorders, such as Cri-du-Chat syndrome are caused
by this type of mutation

Unless these mutations occur in multiples of three, they cause a frameshift
mutation. All the nucleotides downstream of the deletion or insertion will be
improperly grouped into codons. The result will be extensive missense, ending
sooner or later in nonsense - premature termination.

However, 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 incase of a
mutation

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
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)
33
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D.
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)
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



34