Module IV.

Expression of the
genetic information
Unit 11: Transcription.

gr 5/4 12:14
Transcription is the process by which RNA is synthesized using DNA as a
template. Thus, the genetic information stored in the DNA is transferred to the
RNA and, in structural genes, it becomes available for the synthesis of proteins
in the ribosomes. It is a selective process, only the necessary genetic messages
are transcribed at the same time according to the energy needs of the cell.

1
Differences between gene expression of Prokaryotes and Eukaryotes
gr 1) Chromatin structure of eukaryotic DNA.

2) The primary transcription product of the eukaryotic structural genes must be

matured.
more than
3) Monocistronic eukaryotic mRNA vs polycistronic prokaryotic mRNA. one message
4) Transcription and translation coupled in prokaryotes vs compartmentalization
(transcribed / matured / exported / translated) in eukaryotes.

5) Single prokaryotic RNA polymerase vs three eukaryotic RNA polymerases.

pro eu

Messenger RNA
gr 6:20 Prokaryotes Eukaryotes

Three RNA
Polymerases
One RNA Polymerase

Processing

5’ Cap

Translation

Exit to
citoplasm

Translation

Polycistronic mRNA

Monocistronic mRNA
obtaining just one protein

2
Coding and template strands
+1 fixed starting proint

(T = U)

Upstream Downstream
from +1 to -1, -2... from +1 to the end of
the transcriptional unit

Transcriptional units: Promoter, coding región and terminator

Where the transcription begins and ends

transcriptional part from the
transcriptional unit

3
Prokaryotic promoter

40-50 bp opening point

Conserved
distance:
cis signal
17 (+/- 1) pb

-35: Frequency of -10: Transcription

transcription fidelity (copies always easy to denature, easy to
(No. of copies per RNA Pol start at +1) open due to the presence of
complex entry) TATA- or Pribnow-box. T and A

Closed complex Open complex

Transcription in Prokaryotes

multimeric enzyme

RNAP complex
Template read 3’-5’
350-380 KDa Core + 70 KDa s RNA synthesis 5’-3’

E. coli RNA polymerase subunits

4
Transcription in Prokaryotes
27:39
Closed-Open complex
Closed promoter
RNA polymerase
complex

s subunit
Open promoter
complex
Open RNAPol
complex protects
DNA from -40 to
+3 (+/-3)

35:03

Helicases and
Topoisomerases

Iniciation,
elongation and
termination

5
Prokaryotic terminator
rho-independent and rho-dependent.
Conserved distance
(40 pb) A and B: inverted repeats
A B

rho-dependent termination

rho: homohexamer with ATP-dependent helicase activity that

interacts with the DNA-RNA hybrid, pulling apart DNA and
RNA and thus releasing the transcript.

Transcription control in prokaryotes

Francois Jacob Jacques Monod

6
gr Transcription control in prokaryotes

Alternative
s factors

Lac Operon

z codes for b-galactosidase: it catalyzes the hydrolysis of lactose into glucose and galactose.
y codes for galactoside permease: transports b-galactosides into the bacterial cell.
a codes for thiogalactoside transacetylase: catalyzes the transfer of the acetyl group from
acetyl Coenzyme-A to 6-OH from a thiogalatoside acceptor. It is not essential for the
metabolism of lactose, but it is necessary to detoxify other products that enter by permease.

control center: promoter + operator sequence

Lactose Operon
De- Regulator Control Structural
repressor gene elements genes

Lactose

Repressor Promoter b-galactosidase Transacetylase

protein
Operator Permease

Gene arrangement on bacterial chromosome

Regulator Control Structural
gene elements genes

DNA

7
Lactose Operon W/O de-repressor
regulator gene RNA polymerase
DNA

Repressor
Transcription binds
operator
mRNA

Translation
No transcription
Active There is no gene transcription
repressor because repressor is blocking RNA
protein polymerase accces to promoter

9:36
Lactose Operon W/ de-repressor
RNA polymerase
DNA

Transcription Transcription
polycistronic
mRNA messenger

Translation Translation
Lactose mRNA

Protein folding

Active
repressor
protein

b-galactosidase Permease Transacetylase

8
Transcription control in prokaryotes: lac Operon

Tryptophan Operon
Regulator Control Structural
Co-repressor gene elements genes

Tryptophan

Repressor Promoter
protein Operator

Gene arrangement on bacterial chromosome

Regulator Control Structural
gene elements genes

DNA

Active when there is need to synthesize tryptophan.

Negative control mediated by corepressor (tryptophan).

9
Tryptophan Operon W/O co-repressor
RNA polymerase
DNA

Transcription Active repressor Transcription

protein

mRNA

Translation Translation
mRNA

Protein folding

Active
repressor
protein

Repressor without co-repressor could not join the operating region.

Tryptophan Operon With co-repressor

RNA polymerase
DNA
Repressor
Transcription binds
operator

mRNA
Active
repressor
Translation protein

No transcription
Active There is no gene transcription
repressor because co-repressor bind to
protein repressor protein allows this to
enter the operator, blocking RNA
Tryptophan polymerase accces to promoter

10
Repression/Activation. Ara Operon (E. coli)

1.- Repression at
low concentration
of arabinose

2.- Induction at activator when there is arabinose

high concentration
of arabinose

Regulation of gene expression in eukaryotes

1.- Reversible: Similar to bacterial;

Genes that are regulated according
to metabolic needs.

2.- Non-reversible: differentiation

genes; in development processes
there is growth but also
differentiation, where the cell is no
longer able to “go back" in its genetic
program.

11
RNA and RNA polymerases en eukaryotes

Eukaryotic transcription complex

Eukaryotic transcription complex covering basal promoter

12
Eukaryotic promoter

-cis elements:
DNA sequences
(response elements)
-trans elements:
proteins
(transcription factors)

Control of transcription in eukaryotes

General
transcription
factor

Upstream
factor

Binding to double strand DNA through the T ATA box,

twisting and opening the helix.

Binding to its cis-signal (GC-Box) through the

Zn-finger motif.

The most typical structural motifs shared by

different transcription factors to interact with
DNA are 1.-the helix-alpha-helix motif
(homeobox; 60 aa), 2.-the zinc finger motif
Binding of its
Inducible (consensus Cys-X(2a4)-Cys-X3-Phe-X3-Leu-
cis-signal
X2-His-X3-His or else Cys-X2-Cys-X13-Cys-
factor though a four
X2-Cys for steroid receptors), 3.-the leucine
Zn-fingers
zipper motif (LeuZ) and 4.-the amphipathic
motif.
DNA- and protein-binding motif helix-loop-
helix (HLH).

13
gr General transcription factors:

•TFIID: 2 subunits (TBP and 1

complex of 10 TAFs)
•TFIIB: (1 protein) mediates the
interaction between TFIID and
Polymerase
•TFIIF: (4 proteins) stabilizes the
DNA-TBP-TFIIB complex.
•TFIIE: (4 proteins) regulates
TFIIH and together promote
formation the addition of the first
nucleotide of RNA.
•TFIIH: is the largest complex (9
subunits) helicase activity and DNA
repair.

RNA polymerase (12 subunits) requires

these factors to:
CTD domain
1.-To place the enzyme correctly on DNA
2.- To regulate its own activity

14
2:29

RNApol transcribes beyond the coding sequences of most genes. Transcription ends when
the RNA polymerase finds a stop sequence in the DNA. In eukaryotes, the termination
sequence in the template strand is TTATTT, which is transcribed to the pre-mRNA as
AAUAAA. Thus, cleavage occurs after this signal at the 3 'end of the pre-mRNA while the
RNApol continues to transcribe. The Rat1 endonuclease binds to the 5 'end of the RNA
tail, moves towards the RNApol and degrades the RNA as it proceeds in the 5' → 3‘
direction. When Rat1 reaches the polymerase, transcription is stopped.

Termination of transcription in eukaryotes

15
Synthesis and processing of hnRNAs: splicing mechanism

Synthesis and processing of hnRNAs: splicing mechanism

Small nuclear RNA

16
Splice variants by alternative splicing mechanism

15:00 aprox

“An alternate splice acceptor site in the 4th exon of the glucokinase gene was identified in two
glucokinase cDNAs from rat insulinoma tissue. Use of the alternate splice acceptor site
results in a 51-nucleotide in frame deletion in the beta cell glucokinase mRNA and
removal of 17 amino acids from a region of the protein situated between the putative
glucose and ATP binding domains. Analysis of the pattern of RNA splicing in tissues
containing beta cells indicates that the splice acceptor site utilized in producing hepatic
glucokinase mRNA is also utilized in the beta cell”.
Magnuson and Shelton, J Biol Chem. 1989 Sep 25;264(27):15936-42

Alternative promoters
(Glucokinase gene example)

“An alternate promoter in the glucokinase gene is active in the beta cell and produces a
glucokinase mRNA which is longer and that has a different leader sequence and translation
start site than the hepatic glucokinase mRNA. The glucokinase beta cell promoter is located
at least 12 kilobases upstream from the glucokinase hepatic promoter. Transcription from
the glucokinase beta cell promoter initiates over a region of 62 bases. The absence of a
TATA box homology in the proximal promoter region may account for the diffuse
transcriptional initiation. Translation of the beta cell glucokinase mRNA predicts a
glucokinase isozyme that is different from the hepatic isozyme by 15 amino acids at
the N terminus. The use of alternative promoters apparently enables the glucokinase gene
to be regulated by insulin in the liver and by glucose in the beta cell, thus possibly
constituting an important feedback control loop for maintaining glucose homeostasis”.

Magnuson and Shelton, J Biol Chem. 1989 Sep 25;264(27):15936-42

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