SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
DNA Transcription
“ 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;

today 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
Intro Molecular Cell Biology (BIOL 210); 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
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.

The graphic below shows the path of genetic information from the DNA molecule, via
the messenger RNA molecule over to the ribosome
The way from a gene to a cellular protein

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 called helicases help to unwind and open the DNA double helix
at distinct places and create single stranded DNA regions
- the distinct places along the DNA strand where transcription begins are called
transcription start sites (see Graphic below)
- at the transcription start site, a complex protein cluster, the so-called
transcriptome, forms; in eukaryotic cells, the transcriptome is comprised of
many protein components

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)
3
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Genes & Transcription start sites
Transcription start site
gene
I. Mammalia
II. Yeast (S. cerevisiea)
gene
GpC box

the polymerization of the new nucleotides during the process of DNA transcription is
catalyzed by an enzyme called RNA polymerase (see 3D computer image of the
yeast RNA polymerase II below)
- RNA POL II is the central enzyme of gene expression in eukaryotes
- The enzyme is comprised of 10 sub-units (Rbp 1,2,3,5,6,8,9,10,11,12), which
all are necessary for successful gene transcription into a mRNA molecule
- RNA POL II is an evolutionary highly conserved enzyme which contains the
ions Mg2+ and Zn2+ as important co-factors and shows a typical DNA binding
groove

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 DNA transcription process
- In order to begin transcription, RNA polymerase requires a number of so-called
transcription factors (TFs), e.g. TFIIA, TFIIB, etc.
- 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 (see Graphic above)
- 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
4
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Computer-assisted image of RNA Polymerase II (= RNA POL II) of yeast
(based on X-Ray crystallographic data)
Top View
“Active Site”
DNA
mRNA +
pore
8
Mg2+
adapted with modifications from: Cramer P., et al., Science 288 (5466): 640-640 (2000)

while prokaryotic cells, e.g. bacteria, only have one type of RNA polymerase (POL),
which is responsible for gene transcription, there are three different types of RNA
polymerases (= RNA polymerase I, II & III) found in eukaryotic cells (see Figure
below)
- the bacterial RNA polymerase is a tetrameric protein complex made up from one
β subunit, one β’ subunit and two α subunits, which form the functional bacterial
ββ’αα RNA POL complex
- the three eukaryotic tetrameric RNA polymerase complexes are more
complicated in its composition and are made up from one large L‘ subunit, one L
subunit, two different small molecular weight α-like subunits and a series (4 – 7)
of associated small molecular weight proteins
5
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Different RNA polymerases in bacteria and eukaryotic cells
Molecular Weights
160 -220 kDa
128 - 150 kDa
Bacteria
(E.coli)
Eukaryotic cells
44 kDa
19 & 40 kDa
10 - 27 kDa
 all RNA POL subunits are necessary for optimum
eukaryotic RNA polymerase function

DNA transcription is a highly coordinated and regulated cellular process and both,
prokaryotic and eukaryotic cells, have components and mechanisms to turn genes
on or off
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SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.

DNA transcription and gene expression depends on diverse factors, such as:
1. environmental/nutritional factors
2. the presence of hormones or growth factors
3. the presence of pathogens, e.g. fungi, viruses, bacteria
the cells of living organisms can regulate expression of genes by many means;
one of it is by regulation of gene transcription

regulation of gene transcription requires:
1. RNA polymerase
2. DNA-binding proteins
 transcription factors, repressors, activators
 bind to gene regulatory sequences
3. Regulatory DNA sequences associated with genes
 docking sites for transcription factors

our modern understanding of transcriptional regulation was pioneered by the French
scientists F. Jacob and J. Monod in the 1960s who carefully studied gene
expression in the symbiotic intestinal bacterium Escherichia coli (E.coli)

they developed the “repressor-operon model” of transcriptional regulation working in
procaryotes (“lac operon” of E.coli)
- the lac operon contains the three genes Z, Y and A, a transcription control region
and the gene for a transcriptional repressor protein
- the Z, Y, A genes of the lac operon are rapidly induced and expressed when E.
coli has to grow in a medium containing the disaccharide lactose as the only
carbon source  all three genes of the lac operon are coordinately regulated
(for more details: see  “Gene Regulation” section of this website)

prokaryotic transcription starts with the binding of the enzyme RNA polymerase
(POL) to its promoter sequence up- stream of the transcription start site

in the bacterial lac operon (see separate website section ), efficient DNA binding
requires high affinity of the polymerase for the lac promoter and the presence of the
transcription activator protein cAMP-CAP
- cAMP-CAP and RNA polymerase cooperatively stimulate each other's DNA
binding
- mutant cAMP-CAP in certain E.colis strains does not show transactivation and
fails to activate lac transcription

transcriptional regulators, e.g. cAMP-CAP, can activate transcription from different
promoters, e.g. of the lac operon and the gal operon
- e.g. the trp operon repressor also suppresses transcription of the single-gene
operon aroH
- transcription from some promoters is initiated by alternative POL sigma (σ)
factors, which recognize different consensus promoter sequences

many bacterial responses are controlled by two-component regulatory systems
- e.g. control of the E. coli glnA gene by NtrC and NtrB
7
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
- e.g. the E.coli PhoR/PhoB phosphate regulatory system

in eukaryotic cells, gene expression and DNA transcription is mostly regulated by:
1. cell-cell interactions
2. hormones and growth factors
3. environmental factors (less frequent)
- gene control in metazoans requires the precise execution of multiple genes
during embryonic development to orchestrate the morphogenesis into the adult
form
- most genes in the different cell types of higher Eukaryotes are regulated by
controlling their transcription
- we observe differential synthesis of organ or tissue-specific proteins due to
differential transcriptional regulation
- regulation of transcriptional activity in eukaryotic cells requires:
1. “cis-acting”, regulatory DNA sequences
 often many kilo-bp apart within the genome
 regulatory transcription control elements often identified with the
help of gene reporter plasmids (see section below)
2. promoter sequences
3. transcription factors
 transcription factors are proteins with DNA-binding and proteinprotein interaction domains (see section below)

DNA transcription in eukaryotes is executed by three different RNA polymerases,
designated POL I, II and III
1. POL I
- is located in the nucleolus
- synthesizes pre-rRNA, which is processed into 28S, 5.8S, and 18S
rRNAs
2. POL II
- synthesis of tRNAs, 5S rRNA, and many small, stable RNAs (role in
splicing, transport, gene silencing?)
3. POL III
- transcription of mRNAs of all cellular proteins
 all 3 POLs show different sensitivities towards the mushroom poison
α-amanitin (POL II > II > I), which strongly blocks eukaryotic DNA
transcription

The successful guidance and docking of the RNA polymerase to the transcription
start site of a gene is dependent on the presence of “helper proteins”, so-called
transcription factors (TFs)

transcription factors are proteins that can be either activators or repressors or gene
transcription
8
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Experimental Methods
Commonly applied methods for identification and functional study of transcription factors
are DNase I footprinting and electrophoretic mobility shift (EMSA) assaying
 both methods rely on the use of labeled DNA fragments of identified regulatory DNA
sequences
 examples of identified eukaryotic transcription factors are:
1. GAL4 (yeast)
- important TF for expression of enzymes catalyzing galactose metabolism
2. TFII A,B, E, F, H
3. myc
4. AP-1 (c-jun/fos)
5. NFκB
6. Sp1
7. Forkhead

all transcription factors contain two characteristic functional domains (see Figure
below):
1. a DNA-binding domain (DBD)
 interacts with specific DNA sequences
2. a trans-activation domain (AD)
 interacts with other promoter-associated proteins
Functional domains of eukaryotic transcription factors
DBD
AD
Flexible region
Examples of important transcription factors
Gene
GAL4
GEN4
Ubx
Antp
CREB
RARγ
RARα
WT-1
Jun/fos (AP-1)
c-fos
c-myc
p53
Species
yeast
yeast
Drosophila
Drosophila
Mammalia
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H.sapiens
“
H. sapiens
Regulation of
Galactose metabolism
wing formation
wing formation
cAMP-response/PKA substrate
retinoid response genes
retinoid response genes
fct.? (Wilm’s tumor)
growth-promoting genes
cell cycle protein genes
cell cycle protein genes
genes for CKIs
• many transcription factors are over-expressed or mutated in forms of cancer
9
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.

eukaryotic transcription factors (TFs) contain a variety of structural motifs that
interact with specific DNA sequences = DNA-binding domains (see Figure below)
1. Homeodomain (“homeobox”) proteins
- they play an important role in embryonic development
- e.g. Antp, Ubx (Drosophila)
2. Zinc-Finger domain/proteins
- TF with the amino acid consensus sequence Tyr/Phe-X-Cys-X2–4-Cys-X3-Phe/
Tyr-X5-Leu-X2-His-X3–4-His
- this transcription factor domain binds one zinc ion
- it is the most common DNA-binding motifs in eukaryotic TFs

successful binding and exact “docking” of the RNA polymerase to the transcription
start site requires the presence of transcription control sequences and so-called
“cis-acting” regulatory DNA sequences located on the DNA strand

following transcription control sequences that regulate transcription of eukaryotic
protein-coding genes are known:
1. TATA box
 located ≈ 25–35 base pairs upstream of the transcription start site
 important for the exact positioning of the RNA polymerase
 point mutations in that region drastically affect the transcription rate
 many “housekeeping genes” do not contain a TATA box or an initiator
2. Initiators
 are alternative promoter elements of some eukaryotic genes
 they have a C at the −1 position and an A at the +1 position of the
transcription-start site
3. CpG islands
 are CG-rich stretches of 20 – 50 nucleotides ≈ 100 base
pairs upstream of the transcription start-site region
 often seen in genes of house keeping proteins
 binding site of the SP1 transcription factor
 region is susceptible to the HpaII restriction enzyme
4. Promoter-proximal elements
 lay 100 – 200 base pairs upstream of the transcription start site
 mutational insertions of 30-50 bp between promoter
proximal element and TATA box decrease transcriptional rate
5. Enhancers
 located thousands of bp away from the start site
 can be located:
- upstream from a promoter
- downstream from a promoter
- within an intron of a gene
- downstream from the final exon of a gene
 many enhancers are cell-type specific
 e.g. 100-bp enhancer DNA sequence of SV40
10
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.

upon cell stimulation, activated transcription factors together with the RNA
polymerase begin to assemble the transcription-initiation complex at the gene
promoter region (see Graphic below)

the eukaryotic transcription-initiation complex form as a prerequisite of active RNA
synthesis

eukaryotic transcription-initiation complexes include following molecular
components:
1. RNA polymerase II (= POLII)
2. σ70 POL sub-unit (= initiation factor)
3. TBP (= TATA box-binding protein)
 highly conserved C-term. Domain
 binds to TATA box
4. Transcription factor TFII F ( α2β2 tetramer protein)
5. Transcription factor TFII E ( α2β2 tetramer)
6. Transcription factor TFII H
 multimeric protein, consists of 9 protein sub-units
 has ATP-dependent helicase activity
 phosphorylates the C-terminal domain (= CTD) sub-unit of POLII
7. Transcription factor TFII D
 comprises of 11 TAF proteins
 interaction with other proteins, e.g. Sp1
8. Transcription factor Sp1
 binds to the GpC islands/boxes of the promoter region
 transactivation of general initiation complex
9. pppNTPs, ATP

some sub-units of the transcription-initiation complexes play other roles in the cell,
such as
1. activation of cell-cycle kinases & regulation of entry into the S-phase
2. DNA excision repair pathway
 e.g. the TFII H complex
 mutated TFII H protein is observed in patients with the diseases
Xeroderma pigmentosum, trichothio-dystrophy, and Cockayne syndrome

after successful docking of the RNA polymerase to the transcription start site of a
gene and functional assembly of the transcriptome, the RNA polymerase reads
only the so-called sense strand of the double-stranded DNA molecule of the gene
- 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
11
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Sequential assembly of the eukaryotic
transcription initiation complex
TATA box
Gene
12
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Overview: The first events of cellular DNA transcription
Promoter region
transcription
start site

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
13
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
The 3 steps of DNA transcription

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

the activated RNA polymerase recognizes the enhancer and promoter regions
on the DNA and starts adding new nucleotides along the single-stranded ‘senseDNA strand’ following the Watson-Crick base-pairing rule
- since the RNA polymerase can only add new nucleotides to the free 3’hydroxyl group of the last nucleotide, the newly forming messenger RNA
molecule becomes longer in a defined “5’  3’ direction

this step is a crucial step during DNA transcription and involves many 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; the mRNA molecule becomes longer in 5’ 
3’ direction

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
33.. TTeerrm
miinnaattiioonn

the RNA polymerase reaches a sequence of bases, the so-called terminator; as
a consequence the RNA polymerase detaches from the newly formed RNA
molecule and the corresponding gene
- termination is an enormously important part of DNA transcription, since
premature stopping of transcription along the gene would produce truncated
and defective mRNA molecules
14
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
The molecular biology of transcriptional termination
Until recently, information about the exact termination mechanism with which
eukaryotic cells terminate DNA transcription was very scare and vastly enigmatic
since no real “stop sequences” have been found. In 2004, David Tollervey and coworkers (D. Tollervey, et al.; Nature 432: 456-457, 2004) introduced the
“torpedoing concept” according to which the transcribing RNA polymerase is
“torpedo-like” attacked by a series of proteins and the mRNA is digested by a trailing
exonuclease called Rat 1 while it is still synthesized by the RNA polymerase.
According to this exciting termination model, DNA transcription ends when the
exonuclease Rat1 has caught up with the transcribing RNA polymerase .
Successful formation of a mRNA depends on an intact poly-A site formed by the
enzyme PAP. Following proteins have been reported to be necessary for successful
termination of DNA transcription:
1. RH 103
- protein that binds to the C-terminal domain (CTD) of the RNA polymerase
- recruits other proteins, most importantly Rai 1
2. Rai 1
- CTD-binding protein that recruits the exonuclease Rat 1
3. Rat 1
- exonuclease that digests mRNA in 5’  3’ direction (= follows the RNA
POL)
- also ceaves the poly-A site of the newly formed mRNA

3 different kinds of RNA molecules are assembled (= polymerized) by the
transcription mechanism described above
1. messenger RNA (= mRNA)
2. transfer RNA (= tRNA)
3. ribosomal RNA (= rRNA)

all three play a crucial role in the final cellular scenario on the way from the gene
to a functional protein or enzyme
1. The messenger RNA (= mRNA)

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

newly synthesized mRNA – or so-called pre-pro-mRNA - is prone to several
important modifications in order to assure its stability and to control its half-life
within the cell; the mRNA modification which we will look up in more detail are
summarized as RNA processing steps

3 major mRNA processing steps have been identified in cells (see Graphics
below):
15
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
1. CAPing of the 5’-end of the pre-pro-mRNA
- capping is the covalent attachment of a chemical group (=
m7GpppNmpN) with the help of a transcriptome-recruited capping
enzyme and a methyl transferase enzyme (see first Graphic below)
- occurs very early at the beginning of elongation of DNA transcription
2. Attachment of a poly-adenine (= poly-A) tail to the 3’-end of the pre-promRNA molecule
- the polymerization of >200 adenine residues to the 3’-end of the
nascent mRNA molecule (see red-colored circle in the Graphic below)
is catalyzed by an enzyme called Poly-A polymerase (PAP) and
requires a series of helper proteins, such as CPSF, CSIF, CFI
- the process which requires ATP is called poly-adenylation
3. The cutting out of the intron regions of transcribed gene in a cellular
process called splicing
- during RNA splicing, the non-coding 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
- two different splicing mechanisms have been identified in cells (see
Graphic below):
1. a protein-independent self-splicing mechanism
 requires catalytically active snRNA
2. a spliceosome-dependent splicing mechanism
 requires several RNA and protein components, e.g.
U1, U2, U4, U5, U6 snRNA and hnRNPs
 most introns are spliced by this mechanism
Exon - intron organization of genes in eukaryotic cells
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 non-coding DNA, the socalled introns
16
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Summary: The Intron-Exon structure of eukaryotic DNA

in eeuukkaarryyoottiicc cceellllss only a shortened version or a so-called spliced form of
the original RNA strand (= nRNA or pre-pro-mRNA) reaches the ribosome as
mRNA

during DNA transcription into RNA, the eukaryotic cell first transcribes all of
the DNA, including the exons and introns of the gene, into a complementary
RNA copy; in eukaryotic cells, the completely processed – now referred to –
mRNA, leaves the cell nucleus via the nuclear openings or pores
17
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Steps of mRNA processing: CAPing
RNA POL II
DNA
Phosphorylated CTD
mRNA
5’
Capping enzyme
Capping enzyme
Capping enzyme
Methyl transferase
Methyl transferase
mRNA
“CAP”
18
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Steps of mRNA processing
 The capped 5’-end of eukaryotic
Methyl-group
(“CAP”)
= m7GpppNmpN
mRNA
19
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Steps of eukaryotic RNA processing: Polyadenylation
“CAPing”
Nascent (pre-pro) RNA
AUG
5’-CAP
•
RNA-binding
domain (RBD)
- “RGG” box
Proteininteraction
domain
•
3’
A1, A2, C, D, B1, K
Association of
ribonuclear proteins
(hnRNPs)
A1, C, D
5’-CAP
3’
Pyr-rich
3’-ends of introns
1. CPSF, CSIF, CFI
2. Endonuclease
assembly
GU
AAUAAA
5’-CAP
3’
3’- end cleavage
5’-CAP
3. Poly-A polymerase
(PAP)
3’
ATP
Polyadenylation
poly-A
200 – 250
A residues
pro-mRNA
5’-CAP
AAAAAAn 3’
Graphics©E.Schmid/SWC2002

both CAPing & poly-adenylation of pre-pro-mRNA help to prevent the
otherwise very rapid degradation of the otherwise very ‘fragile’ mRNA
molecule by cellular RNA-digesting enzymes called RNAses
20
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Scheme: Eukaryotic RNA Splicing

Splicing of mRNA occurs in eukaryotic cells, but not in bacteria; it is one of the
amazing mechanisms with which eukaryotic cells can form alternative – shorter
or longer – version of mRNA molecules due to removal (splicing out) of different
introns from identical pro-mRNA molecules  differential splicing

Differential splicing creates different transcript version of the same gene and
significantly contributes to an increased arsenal of gene products with an limited
amount of genes within the chromosomal material of an organism
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SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Molecular biology of splicing
Exon 2
5’
Intron 2
GU
Exon 3
3’
AG
Pyr-rich domain
Formation of
Protein-independent
“Self-splicing”
“Spliceosome”
OR
Catalytically active
snRNA
U1, U2, U4, U5, U6 snRNA
5 hnRNPs
Spliced
Intron 2
- Group I introns
- Group II introns
- most introns
(mtDNA, cpDNA)
mRNA
Exon 1
Exon 2
5’- CAP
AUG

Exon 4
(AAAAA)n – 3’
Nuclear Export
5’ - CAP
Exon 3
Nuclear Pore Complex (NPC)
(> 60kDa active transport)
polyA – 3’
in eukaryotic cells, the information-less (= non-coding) introns are removed
from the pre-pro-mRNA by a process called RNA splicing; this edited ( or
spliced RNA) sequence is called mRNA
22
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.

the thus processed and spliced final version of the mRNA travels from the
nucleus into the cytosol to the ribosomes, the place of cellular protein synthesis
- in eukaryotic cells the ribosomes are located on the surface of the rough
endoplasmic reticulum (rER)
- in bacteria they swim freely in the cytosol
Splicing of eukaryotic, but not prokaryotic RNA
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SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); 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
24
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); 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
25
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); 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 involvement of many protein components and the complexity of the
transcriptome, including the RNA polymerase enzyme, makes DNA transcription
vulnerable to the attack by many interfering compounds; many inhibitors of DNA
transcription have been identified by scientists some of which are listed in the
table below
- many of these DNA transcription inhibitors are poisons long known to
humankind, such as the extremely dangerous mushroom poison alphaamanitin
- many bacterial DNA transcription inhibitors are known antibiotics which play
an important role in modern health care
Inhibitors & Poisons of DNA Transcription
- Many chemically synthesized and naturally occuring
molecules are known to interfere with DNA transcription
Molecule
Mode of Action
•
Actinomycin D
- antibiotic; blocks elongation of bacterial RNA
polymerase
•
α/β- Amanitin
- fungal toxins; binds to and inhibits eukaryotic RNA
POL II

5,6- Dichlorobenzimidazole - leads to premature termination of RNA POL
1-β-D-ribofuranoside
•
Distamycin
- inhibits initiation of DNA transcription
•
Rifampicin
- antibiotic; inhibits initiation of DNA transcription
- binds to β-subunit of RNA polymerase
26
SAN DIEGO MESA COLLEGE
SCHOOL OF NATURAL SCIENCES
Intro Molecular Cell Biology (BIOL 210); Instructor: Elmar Schmid, Ph.D.
Inhibitors of DNA Transcription: Amanitin
Amanita phalloides
“Death angel mushroom”
(Basidiomycota)
 extremely poisonous to humans!!
- produces several bicyclic amatoxins
( all are octa-peptides)
α-Amanitin
 Amanitin inhibits the eukaryotic RNA polymerase II
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