Transcription

and
The Control of Gene Regulation
 Learning Objectives

Student can
 Explain both the prokaryotic and
eukaryotic transcription machineries
 Define the post transcriptional
modifications observed in eukaryotes
 Determine the splicing machinery
 Explain different gene regulation
processes in eukaryotes
 Classify different RNA types and their
functions in gene regulation
• DNA cannot directly be used as a template for
protein synthesis.
• Instead, it codes an RNA transcript(mRNA) which
eventually turns into protein.
• Transcription,in general, is coding of DNA into RNA
to lead to mRNA, rRNA and tRNA molecules
 RNA polymerase is the enzyme
responsible for mRNA
transcription.

 Using DNA as the template,it

transcribes the information
present within the DNA.

 It does not need any primer unlike

DNA polymerase in replication
process
 PROKARYOTIC TRANSCRIPTION

• In prokaryotes only one polymerase is present

and is responsible for the synthesis of all
mRNA, rRNA and tRNAs
 PROKARYOTIC TRANSCRIPTION
RNA Polymerase
• İs a complex enzyme made up of many

polypeptides.

• Prokaryotic RNA Polymerase is

composed of α, β, β’ ω and σ

subunits.

• σ (sigma) subunit binds to the

complex a bit weaker.

• As the RNA polymerase synthesizes

almost 10 nucleotides σ subunit

dissociates from the complex

PROKARYOTIC TRANSCRIPTION

RNA Polymerase

 Composed of two α, one β, one β’ and one ω (omega) subunits,

 Core RNA polymerase is responsible for RNA elongation.

 Core RNA polymerase is responsible for catalyzing of NTP

addition.

 For this catalyzing reaction, σ factor is not needed.

 PROKARYOTIC TRANSCRIPTION

Necessities for transcription,

DNA template,
RNA polymerase enzyme,
Ribonucleotide triphosphates(ATP, GTP, CTP,
UTP) and
Magnesium ion
 Transcription begins with the binding of RNA core enzyme
to the promotor region (In prokaryotes-The nucleotides
downstream of 10 to 35 Shine Dalgarno Sequence)
TranscriptionTermination
Rho-dependent
As a helicase rho
recognizes the hairpin
structure of GC rich
regions and leads to
dissociation of RNA
polymerase from mRNA

Rho-independent
Here the termination
sequence is leaded to
RNA polymerase
dissociation from the
complex
 RNA Types
 Mesenger RNAs (mRNA): as a template for protein synthesis

 Ribosomal RNAs (rRNA ) and Tranporter RNAs (tRNA) : translating

the code in mRNA into protein

 Small RNAs
– mRNA splicing
– rRNA processing etc.
– Regulation of gene
expression
 mRNA is messenger RNA which carries the
information of DNA to the cytosol to be
converted into protein.

 rRNA is ribosomal RNA which is used during

ribosome biosynthesis to get a mature
ribosome.

 tRNA is transfer RNA present within the

cytosol that brings aminoacids to the ribosome
according to anticodon it has,with respect to
the codon present on the mRNA.
 Every transcribed segment of DNA is called transcription unit.

 In prokaryotes, instead, a series of adjacent genes are

transcribed altogether (polycistronic).

 In eukaryotes, the transcription unit carries the information

that belongs only to a unique gene (monocistronic).

 EUKARYOTIC TRANSCRIPTION

Promotor Regions of Eukaryotic Genes

Downstream of transcription starting point(+1)

- 80 nucleotide CAAT sequence

- 30 nucleotide TATA sequence

In some genes GC element

Eukaryotic polymerases need transcription factors to

initiate transcription.
 EUKARYOTIC TRANSCRIPTION
Initiation of Transcription
• Transcription initiation begins with the recognition of
promotor regions via RNA Polymerase II and the
transcription factors .
• 5 general transcription factors are needed.
– TF II A,
– TF II B,
– TF II D,
– TF II E,
– TF II F,
– TF II H
 EUKARYOTIC TRANSCRIPTION
Initiation of Transcription
A- TF II D (general transcription factor);

binds to TATA box.

(TF-IID is composed of 10 polypeptides of TBP

(TATA binding protein) and TAF

(TBP associated protein)

B- Then, TF IIB and TF IIA binds to TBP

C- TF IIF with RNA polymerase bind to the

complex

D- Eventually,TF IIE and TF IIH binds and the

initiation complex is ready to go.

 EUKARYOTIC TRANSCRIPTION
Promotor Regions of Eukaryotic Genes

• In front of TATA box, there are 3 sequence elements

enabling efficient transcription;a CCAAT box and two

GC boxes (consensus sequence GGGCGG)

Transcription
Initiation
Elongation
Termination
Post-transcriptional Modifications
 EUKARYOTIC TRANSCRIPTION

RNA Splicing
After transcription,
The introns are removed and
the exons are united via a
process known as Splicing.

The structure that performs

the splicing process is called
spliceosome.

It is the last mode of post-

transcriptional modification
of eukaryotic cell prior to
leaving nucleus.
 EUKARYOTIC TRANSCRIPTION
Post-transcriptional Modifications

 Addition of 7-methyl guanosine cap

 Addition of PolyA tail
 Splicing via spliceosomes
 Translocation from nucleus to
cytosol to be translated into protein
EUKARYOTIC TRANSCRIPTION
The Control of Gene Expression
 A neuron and a liver cell can have different structures
eventhough they have the same genome.
 The thing that makes them different is the levels of the genes
that they have expressed.
 The difference expression patterns of the same gene-Beta-actin in
different cell types
 Differences in the proteins expressed by two human tissues, (A) brain and
(B) liver
The mode of gene regulation
 A cluster of bacterial genes can be transcribed from a single promoter
Genes can be switched off by repressor proteins
Repressors Turn Genes Off and Activators Turn Them On
 The Lac operon encodes proteins
required to import and digest the
disaccharide lactose.

 In the absence of glucose, the

bacterium makes cAMP, which
activates CAP to switch on genes
that allow the cell to utilize
alternative sources of carbon—
including lactose.

 It would be wasteful, however, for

CAP to induce expression of the Lac
operon if lactose itself were not
present.

 Thus, the Lac repressor shuts off

the operon in the absence of lactose.

 This arrangement enables the control

region of the Lac operon to integrate
two different signals, so that the
operon is highly expressed only when
two conditions are met: glucose must
be absent and lactose must be
present.
 Eukaryotic Transcription Regulators Work in Groups
 Transcription activators often exhibit transcriptional synergy, where
several DNA-bound activator proteins working together produce a
transcription rate that is much higher than the sum of their transcription
rates working alone.
 Some ways in which the activity of transcription regulators is controlled inside
eukaryotic cells
 A small set of transcription regulators can convert one differentiated cell
type into another
 Specialized Cell Types Can Be Experimentally Reprogrammed to Become
Pluripotent Stem Cells
 CELL MEMORY

 A positive feedback loop can create cell memory. Protein A is a master

transcription regulator that activates the transcription of its own gene—
as well as other cell-type-specific genes.
 All of the descendants of the original cell will therefore “remember”
that the progenitor cell had experienced a transient signal that initiated
the production of protein A.
Genomic imprinting

The expression of a small

minority of genes depends
on whether they have been
inherited from the mother
or the father: when the
paternally inherited gene
copy is active, the
maternally inherited gene
copy is silent, or vice versa.

This phenomenon is called

genomic imprinting.
Genomic imprinting

 In Angelman syndrome,
a disorder of the nervous
system in humans that
causes reduced mental
ability and severe speech
impairment, results from a
gene deletion on one
chromosomal homolog and
the silencing, by imprinting,
of the intact gene on the
other homolog
X-Inactivation

Mammals achieve dosage

compensation by the
transcriptional inactivation of
one of the two X chromosomes
in female somatic cells, a
process known as X-inactivation.

As a result of X-inactivation,
two X chromosomes can coexist
within the same nucleus, exposed
to the same diffusible
transcription regulators, yet
differ entirely in their
expression
 X-Inactivation
How is an entire chromosome
transcriptionally inactivated?

X-chromosome inactivation is
initiated and spreads from a
single site near the middle of
the X chromosome, the X-
inactivation center (XIC).

Within the XIC is a

transcribed 20,000-
nucleotide lncRNA (called
Xist), which is expressed
solely from the inactive X
chromosome.

Xist RNA spreads from the XIC over the entire chromosome and directs
gene silencing.

Although we do not know exactly how this is accomplished, it likely involves

recruitment of histone-modifying enzymes and other proteins to form a
repressive form of chromatin
X-Inactivation
POST-TRANSCRIPTIONAL CONTROLS

The post-transcriptional
controls, which operate
after RNA polymerase
has bound to the gene’s
promoter and has begun
RNA synthesis, are
crucial for the regulation
of many genes.
POST-TRANSCRIPTIONAL CONTROLS
 Riboswitches probably represent ancient forms of gene
control.
We discussed the idea that, before modern cells arose on
Earth, RNA played the role of both DNA and proteins, both
storing hereditary information and catalyzing chemical
reactions («the Evolution of the cell» lecture)
POST-TRANSCRIPTIONAL CONTROLS
Two mechanisms of eukaryotic mRNA
decay
POST-TRANSCRIPTIONAL CONTROLS
Two mechanisms of eukaryotic mRNA
decay
 Gene Regulation via Regulators

 50 years ago, Jacob and Monod discovered «the

repressors» in their gene regulation studies.
 They were not able to conclude whether these repressors
are RNAs or proteins.
 In that case,they pointed out that these repressors were
to be RNAs.
 Because in many case «the regulators» are known to be
the proteins that bind to DNA and the clues fo these
repressors to be accepted as proteins led this idea to be
forgotten eventually.
 Gene Regulation via Regulators

 The number of studies on RNA regulators have

increased in recent years.
 Especially, RNA regulators which function in
transcriptional and translational levels of gene
regulation.
This new window opened via 2 sources:
1- The discovery of the microRNAs (early 1990’s)
2-The discovery of RNA interference ( late 1990’s)
 RNA interference (RNAi)
• A various small RNAs repress or silence the
genes that have homologous regions with their
own sequences.
• This type of silencing is called RNA
interference(RNAi).
• RNAi is observed during:
-Inhibition of mRNA translation
-Facilitating mRNA degradation
-Silencing of the promotor responsible for mRNA
expression
 RNA interference (RNAi)
 RNA interference is a basic mechanism in
gene expression regulation.
 In some eukaryotic gene transcripts,there
are regulatory RNA elements.
 These elements fuction as binding to
regulatory proteins (HIVTAT protein, etc.)
 Nowadays, RNAs are known to have a
significant role in gene regulation.
 Small RNAs
• They are defined in bacteria for the first
time
• Some of them join the replication process of
the plasmid
• The remainings join the regulation of gene
expression.
• miRNA, siRNA, and piRNA
 Small RNAs
Micro RNAs (miRNA)
 They have encoded via RNA transcription in nucleus and
have specific regulatory functions.
 They are 20-22 nt in length and have only one strand

 miRNA genes are usually transcribed via RNA polymerase

II

 To get a mature miRNA, the precursor miRNA should be

excised off through an enzyme called DICER.
To synthesize a mature miRNA

 The primer transcript is transcribed which

can be hundreds of nucleotides in length(pri-
miRNA)
 Drosha enzyme is used to process pri-miRNA
and the previous miRNA(pre-miRNA) that is in
hairpin structure and 70-100 nt in length.

 The translocation to cytoplasm is provided via

exportin 5.
To synthesize a mature miRNA
 In cytoplasm,the pre-miRNA is reprocessed via
DICER enzyme (RNAse III) and si-RNA like duplex
structure is constructed.
 With the help of argonaute and some other
proteins,one of the strand is degraded and the
remaining strand is bind to RISC (RNA induced
silencing complex).
 The miRNA-RISC structure is now ready to silence
the genes of interest.
 Argonaute, also called «the slicer» , is a catalytic
unit which degrades one of the strand of the
miRNA.
 If the sequence of the transcript of interest is
highly complementary to miRNA sequence, the
target transcript is rapidly degraded.
 If the sequence of the transcript of interest is
loosely complementary to miRNA sequence(some
nucleotides are not matched), then the target
transcript is not readily degraded. Instead,the
translation is inhibited.
 As the translation is inhibited or terminated the
transcript of interest is gradually degraded.
 RNAi silincing is very efficient.
 Just a little dsRNA is capable of inducing the
complete silence of the whole gene expressions.
 siRNA
 The presence of double-
stranded RNA in the cell
triggers RNAi by attracting a
protein complex containing
Dicer, the same nuclease that
processes miRNAs
 This protein cleaves the double-
stranded RNA into small
fragments (approximately 23
nucleotide pairs) called small
interfering RNAs (siRNAs).
 Argonaute protein is used as the
slicer.
siRNA
 piRNA
• A third system of RNA interference relies on piRNAs
(piwi-interacting RNAs, named for Piwi, a class of
proteins related to Argonaute).
• piRNAs are made specifically in the germ line, where
they block the movement of transposable elements.
• The processing differs from that for miRNAs and
siRNAs (for one thing, the Dicer enzyme is not
involved), and the resulting piRNAs are slightly longer
than miRNAs and siRNAs.
• They are complexed with Piwi rather than Argonaute
proteins.
 Bacteria Use Small Noncoding RNAs to
Protect Themselves from Viruses
 Many features of this defense mechanism, known as the CRISPR
system, resemble those we saw above for miRNAs and siRNAs, but
there are two important differences:
 First, when bacteria and archaea are first infected by a virus, they
have a mechanism that causes short fragments of that viral DNA to
become integrated into their genomes. These serve as “vaccinations,”
in the sense that they become the templates for producing small
noncoding RNAs known as crRNAs (CRISPR RNAs) that will
thereafter destroy the virus should it reinfect the descendants of
the original cell.
 This aspect of the CRISPR system is similar in principle to adaptive
immunity in mammals, in that the cell carries a record of past
exposures that is used to protect against future exposures.
 The second distinguishing feature of the CRISPR system is that
these crRNAs then become associated with special proteins that
allow them to seek out and destroy double-stranded DNA molecules,
rather than single-stranded RNA molecules
Bacteria Use Small Noncoding RNAs to
Protect Themselves from Viruses
 Transcription
and
The Control of Gene Regulation