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BiochemistryMolecular biology 2.

Transcription

University of Veterinary Medicine

Department of Physiology and Biochemistry

Structure of RNA

• Structure

– Ribose + U

– Single stranded polynucleotide

• BUT: intramolecular base pairing! (tRNA)

Types of RNA

• Types– mRNA (messenger RNA: transcript carrying

genetic information)

– Functional RNA types• tRNA (transfer RNA: carrier of activated amino acids)

• rRNA (ribosomal RNA: component of ribosomes)

• Small RNA types– snRNA (small nuclear RNA: ribozyme catalysing splicing of

mRNA in Eukaryotes)

– snoRNA (small nucleolar RNA: ribozyme catalysing splicing of tRNA, rRNA and snRNA)

– micRNA (mRNA inhibiting complementary RNA: regulation of translation)

– siRNA (small interfering RNA: degradation of RNA molecules)

Function of types of RNA in gene expression

micRNA snRNA

micRNA(mRNA inhibiting complementary)

snRNA(small nuclear)

Transcription

• Transcription = synthesis of RNA from

(template strand of) DNA

– Strands of DNA:

• Coding (sense) strand: stores genetic information

• Non-coding (antisense, template) strand:

complementary to coding strand serves as template

(„pattern”) for transcription

• Genetic information is transcribed from DNA

to RNA

The transcription unit

• The transcription unit (TU) is the

functional unit of DNA

• It is composed of the following regions:

– (1) promoter: regulatory function

– (2) RNA coding region

The structure of transcription unit

Termination signal:

transcription finishes here

The promoter (1)

• It is responsible for the regulation of transcription

• The most important consensus sequences of promoter region are:– TATA-box (core promoter, called Pribnow-box in

Prokaryotes):• Rich in T and A bases

• RNA polymerase binds tightly to this box

– GC-box• Rich in G and C bases

• RNA polymerase binds loosely to this box

– CAP-cAMP binding site (only in Prokaryotes)• Binding of CAP-cAMP complex one of the prerequisites of

transcription (see later)

The transcription unit

• Transcription start site: located between promoter and RNA coding region transcription starts from this point

• „Upstream” direction = from the start site towards promoter („left”), bases found here get number minus 1, 2 etc., when mowing away from start site

• „Downstream” direction = from the start site towards RNA coding region („right”), bases found here get number plus 1, 2 etc., when mowing away from start site

The RNA coding region (2)• At the beginning and at the end of RNA coding region: such DNA

sequence, which is transcribed but is not coding amino acid,

therefore is not taking part in the protein synthesis (translation).

– Abbreviation is: UTR (untranslated region) function in transport and

protection of RNA molecules

• Gene/structural gene: such DNA sequence, which is coding one

RNA molecule (in case of mRNA, it means coding one protein

molecule, see later)

– Prokaryotes: one transcription unit is composed of several genes

polycistronic transcription unit

• The transcription of genes of one TU is regulated commonly

• Allows coarse regulation of transcription (if transcription happens, all the genes

are transcribed)

• Proteins coded on the same TU have function in the same metabolic process

– Eukaryotes: one transcription unit is composed of one gene

monocistronic transcription unit

• Fine, separate regulation of transcription of each gene

The RNA coding region(2)

• In Eukaryotes, introns and exons can be found

inside of genes

– Introns: can be found in pre-mRNA (primary

transcript) only they are cut out mature mRNA

contains no intron

– Exons: mature mRNA contains exons only

Transcription of mRNA of

Prokaryotes

• mRNA transports genetic information from

DNA to the protein synthesis (translation)

• The transcription of mRNA of Prokaryotes has

3 phases:

– Initiation (1)

– Elongation (2)

– Termination (3)

Initiation of transcription of Prokaryotes

(1)

• In Prokaryotes, one single enzyme (DNA-

dependent) RNA polymerase catalyses

transcription of all types of RNA

• RNA polymerase has high affinity to DNA, but

not specifically to the promoter region

– RNA polymerase apoenzyme + sigma (σ)

factor = RNA polymerase holoenzyme

specific affinity of the enzyme to the promoter

holoenzyme binds tightly to TATA- (Pribnow-)

and loosely to GC-box of promoter

Initiation of transcription of Prokaryotes

(1)

• Binding of CAP-cAMP complex to the

binding site on the promoter is necessary

for the initiation of transcription (see later)

Elongation of transcription of

Prokaryotes (2)

• The RNA polymerase builds nucleotides

(complementary to the template strand of DNA)

into mRNA chain mRNA is being

synthesised

– RNA polymerase uses nucleoside triphosphates

(NTPs = ATP, GTP, CTP, UTP) as substrates after

splitting one inorganic pyrophosphate (PPin),

nucleoside monophosphates (NMPs) are build in

mRNA chain

Termination of transcription of

Prokaryotes(3)

• Termination starts at the end of transcription unit, at

termination signal

• Two possibilities of termination exist:

– Rho(ρ)-factor independent termination

• A region, rich in G and C bases can be found on mRNA at the

region of termination signal

• H-bonds formed between G and C bases destabilise DNA-RNA

complex DNA chain, mRNA and RNA polymerase dissociate

– Rho(ρ)-factor dependent termination

• G-C rich region slows down mRNA synthesis

• The so called rho(ρ)-factor follows RNA polymerase enzyme during

transcription

• Rho(ρ)-factor reaches RNA polymerase when it is slowed down

and catalyses dissociation of DNA chain, mRNA and RNA

polymerase (ATP!)

The rho(ρ) -factor independent termination of

transcription of Prokaryotes

G-C rich region

of mRNA forms

a loop

Loop

destabilises

DNA-RNA

complex

The rho(ρ) -factor dependent termination

of transcription of Prokaryotes

RNA polymerase

ρ-factor follows

RNA polymerase

Loop slowes down

RNA polymerase → ρ-

factor reaches the

enzyme

ρ-factor catalyses

dissociation of DNA,

mRNA and enzyme

ρ-factor

Structure of mRNA of

Prokaryotes• Base triplets found on mRNA are called „codons”

• Prokaryotic mRNA is polycistronic: contains transcript

of several genes (no maturation is needed!)

• Between transcripts of genes, so called Shine-

Dalgarno-sequences (RBS = ribosome binding site) are

located during translation, mRNA binds to ribosomes

by these sites

• On the 5’- and 3’-ends: no amino acids are coded (UTR

= untranslated regions) needed for the transport and

protection of mRNA (against RNA degrading enzymes of

cytoplasm)

Regulation of transcription of

Prokaryotes

• Transcription of Prokaryotes is regulated by

operon model

• Operator region: a DNA-sequence on the

promoter or between transcription start site and

UTR binding site for repressor protein

inhibition of transcription

http://www.nature.com/scitable/content/transcription-repression-near-the-promoter-region-14711150


Prokaryotes

• Two prerequisites of transcription:

– No repressor bound on the operator region no

inhibition

– CAP-cAMP complex must be bound to CAP-cAMP

binding site of promoter transcription can start

• CAP = Catabolit Activator Protein

• cAMP = cyclic adenosine-monophosphate an important

regulator („second messenger” molecule of different

metabolic pathways, see next semester)


Prokaryotes

• Two examples for operon model:

– Lactose operon (Lac-operon): regulates

transcription of genes of lactose degrading

enzymes

– Tryptophan operon: regulates transcription

of genes of tryptophan synthesising enzymes

The lactose operon

• Transcription unit codes genes of lactose

degrading enzymes (lactase/β-galactosidase,

permease…)

• Lactose binds to the repressor on the operator

and removes it genes will be transcribed only

when lactose is present (enzyme induction)

• In the presence of glucose, cAMP concentration

decreases CAP-cAMP complex is not formed

and won’t bind to the promoter no transcription

bacteria degrade glucose instead of lactose

The lactose operon

• 4 possibilities exist:Glucose Lactose CAP-cAMP

complex

Repressor Transcription

glucose „+”

less cAMP

complex not bound

lactose „+”

repressor is removed

from operator

glucose „+”

less cAMP

complex not bound

lactose „-”

repressor is bound to

operator

glucose „-”

more cAMP

CAP-cAMP complex

bound

lactose „-”

repressor is bound to

operator

glucose „-”

more cAMP

CAP-cAMP complex

bound

lactose „+”

repressor is removed

from operator

The lactose operon

http://oregonstate.edu/instruction/bi314/fall11/geneexpression.html

The tryptophan operon

• TU codes genes of enzymes catalysing

tryptophan synthesis

• Tryptophan (Trp) acts as corepressor

repressor binds to operator only when activated

by Trp

• When Trp is present: Trp binds and thus

activates repressor inhibition of

transcription bacteria uptake Trp from the

environment

• When Trp is NOT present: repressor is inactive

transcription happens bacteria synthesise Trp

The tryptophan operon

http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/trans/REGULATION.HTML


Eukaryotes

• Similarly to Prokaryotes, mRNA transports genetic

information from DNA to the protein synthesis

(translation)

• Transcription of mRNA of Eukaryotes also has 3

phases (initiation, elongation, termination)

• In transcription, pre-mRNA (primary transcript)

is synthesised first cotranscriptional

processing of mRNA is needed = maturation

of mRNA (pre-mRNA mRNA transformation)


Eukaryotes

• Distinct (DNA dependent) RNA polymerases

catalyse transcription of distinct RNA types:

– RNA polymerase I.: most types of rRNA (ribosomal

RNA) 5.8S rRNA; 18S rRNA; 28S rRNA (rRNA

types will be detailed later)

– RNA polymerase II.: mRNA and types of snRNA

(small nuclear RNA, except: U6 snRNA)

– RNA polymerase III.: tRNA, 5S rRNA and U6 snRNA


Eukaryotes

• RNA polymerases can be characterised

according to the ability of being inhibited

by α-amanitin

– α-amanitin is the toxin of fungus „death

cap” (Amanita phalloides)

• RNA polymerase I.: can not be inhibited

• RNA polymerase II.: inhibition happens

at low concentrations of toxin (enzyme is

very sensitive)

• RNA polymerase III.: inhibition happens

at higher concentrations of toxin

(enzyme is less sensitive)

Initiation of transcription of

Eukaryotes (1)

• Numerous proteins („basal” or „general”

transcription factors) are needed for initiation

• RNA polymerase II (in case of mRNA

synthesis) binds to promoter pre-initiation

complex

• Double stranded DNA uncoils

• First few nucleotides are built in the mRNA

chain

• Basal transcription factors dissociate

initiation complex


Eukaryotes (1)

• Numerous proteins („basal” or „general”

transcription factors) are needed for initiation

1. Binding of TFIID (= transcription factor IID) to

TATA-box

• TFIID is composed of two subunits:

– TBP = TATA-binding protein (recognises TATA-box)

– TAF = TBP-associated factors

2. Binding of TFIIA and TFIIB


Eukaryotes (1)

3. Binding of RNA polymerase II and TFIIF (+

other transcription factors)

4. Binding of TFIIE and TFIIH

• TFIIH is composed of two subunits:

– Helicase

– Protein kinase

Pre-initiation complex is formed

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Eukaryotes

1. Binding of TFIID to

TATA-box

2. Binding of TFIIA and

TFIIB

3. Binding of RNA

polymerase II + TFIIF +

other factors

4. Binding of TFIIE and

TFIIH

Pre-initiation complex


Eukaryotes (1)

5. Helicase subunit of TFIIH uncoils the two

DNA strands

6. The first few nucleotides are built in the

new mRNA chain (NTPs NMPs)

7. Protein kinase subunit of TFIIH

phosphorylises C-terminal domain of RNA

polymerase II. enzyme gets activated by

this phosphorylation

8. Basal transcription factors dissociate elongation starts

6.

7.

8.


Eukaryotes

6. The first few

nucleotides are built in

the new mRNA chain

(NTPs NMPs)

7. Protein kinase subunit

of TFIIH phosphorylises

C-terminal domain of

RNA polymerase II.

enzyme gets activated

by this phosphorylation

8. Basal transcription

factors dissociateC-terminal domain

TRANSCRIPTION

Elongation and termination of

transcription of Eukaryotes (2-3)

• Elongation:

– RNA polymerase II. builds the appropriate

nucleotides in the new mRNA chain, which are

complementary to the template strand of DNA

– Substrates of RNA polymerase II. are nucleosid-

triphosphates (NTPs: ATP, GTP, UTP, CTP)

inorganic pyrophosphate (PPin) is split, and NMPs (AMP,

GMP, UMP, CMP) are built in the new mRNA strand

• Termination:

– Synthesis of new mRNA stops at the end of transcription

unit, at cleavage sequence

Cotranscriptional processing of

mRNA

• During eukaryotic transcription, pre-mRNA is

synthesised first

• The pre-mRNA mRNA transformation is the

so called cotranscriptional mRNA processing =

the maturation of mRNA

– (5’-)capping happens during elongation

– (3’-)tailing = polyadenylation happens during

termination

– Splicing happens during termination


mRNA

• After building approximately 30 nucleotides in

the chain, next step is (5’-)capping:

– A 7-methyl-GTP group (cap) is bound to the 5’-end

of pre-mRNA

– This 7-methyl-GTP cap protects mRNA from RNA

degrading nuclease enzymes and helps its transport

out from the nucleus

The structure of 7-methyl-GTP cap


mRNA

• (3’-)tailing = polyadenylation

– A so called „poly-A-tail” is bound to the 3’-

end of pre-mRNA

• It is composed of 100-200 AMP units

– The poly-A-tail is synthesised by poly-A-

polymerase enzyme

– Tail also protects mRNA and helps its

transport


mRNA - splicing• Splicing: that process in the cell nucleus,

where introns are removed (cut out) and

exons are reunited

– Process is catalysed by snRNAs (= small

nuclear RNAs)

• snRNAs are ribozymes (RNA molecule with enzymatic

property)

– Different types of snRNAs are marked with U1,

U2 etc.

– snRNAs work with proteins

• These proteins are snRNPs (= small nuclear

ribonucleoproteins)

they form spliceosome with pre-mRNA

Schematic picture of splicing


mRNA - splicing

• U1 snRNA binds to 5’-end of intron

phosphodiester bond is split between intron and

exon

• U2 snRNA binds to one AMP (a consensus

sequence) inside the intron

• 5’-end of intron binds to the AMP (as a

„branching point”) inside the intron „intron-

loop” or „intron-lariat” is formed


mRNA - splicing

• U5 snRNA binds to 3’-end of

intron phosphodiester bond is

split between intron and exon intron is removed

• U4 and U6 snRNA are also

needed

– They help the work of the other snRNA

molecules

• The neighbouring exons are

reunited after removal of intron

5’-end of

intron

3’-end of

intron

http://www.phschool.com/science/biology_place/biocoach/transcription/premrna.html


Eukaryotes

• The two most important way of regulation of

eukaryotic transkription:

(1) Modification of structure of chromatins

• Modification of histones (acetylation, methylation,

phosphorylation, etc.)

• Modification of DNA (Methylation)

(2) Regulation with transcription factors

• With the regulation of transcription, gene

expression can be altered

The gene expression

• Gene expression = broadly, the process by

which information from a gene is used in the

synthesis of a functional gene product (usually

protein).

• Gene expression can be influenced and

regulated on the level of…

– Transcription: How many mRNA are produced?

– Translation: How many proteins are produced?

– Post-translation: How many active proteins are

produced?

• In narrow sense, gene expression is the

intensity of transcription

Epigenetic regulation of

transcription

• By changing chromatine structure (histone or DNA

modification)…

– The sequence of nucleotides is not altered

– Transcription and thus gene expression is influenced

– These changes are heritable

This is the so called epigenetic regulation of

transcription ( epigenetics)

• Epigenetically active molecules (from feed or the

environment etc.) are able to influence gene

expression via epigenetic regulation

Histone modifications

Histone modifications

• The structure of histone proteins is covalently

modified leading to the change of structure

of the whole nucleosome influencing the

intensity of transcription (RNA synthesis) regulation of gene expression (see later)

• Possible modifications of histones:– Acetylation: binding of acetyl group

– Methylation: binding of methyl group

– Phosphorylation: binding of phosphate group

– Ubiquitinylation: binding of ubiquitine (= a polypeptide)

– SUMOylation: binding of a protein molecule (SUMO,

structure similar to that of ubiquitine)

Histone acetylation

• Histone acetyltransferase (HAT):

binds one acetyl group to the ε-

amino group of a lysine of the

histone ε-amino group looses one

proton less positive charge

less ionic bonds between histone

and DNA loose, transcriptionally

more active chromatine structure

• Histone deacetylase (HDAC):

removes acetyl group of lysine

positive charge more ionic bonds

tight, transcriptionally inactive

chromatine structure

• HDAC inhibitors (e.g. butyrate,

trichostatin A): histones get

hyperacetylated the affected gene

gets activated gene expression is

stimulated

DNA methylation

• DNA methyltransferase binds methyl group to

a cytosine ( 5-methyl-cytosine is formed) on

the promoter region of DNA transcription is

blocked, the affected gene gets inactivated

gene expression is decreased = „gene

silencing”

Regulation of transcription with

transcription factors

• Cis-regulatory elements:

= such region of the DNA, to where binding of

trans-regulatory element regulates/influences

transcription

– Types:

• Promoter: central role in the regulation of transcription

• Enhancer region: stimulation of transcription

• Silencer region: inhibition of transcription



• Trans-regulatory elements = transcription

factors:

= Proteins, which binds to the appropriate cis-

regulatory element thus transcription gets

regulated/influenced

– Types:

• Basal (general) transcription factors binding to the

promoter prerequisite of transcription (see „Initiation

of transcription”)

• Activators binding to an enhancer region

stimulates transcription

• Repressors binding to a silencer region inhibits transcription



• Review of cis- and trans-regulatory elements:

Cis-regulatory elements

(DNA region)

Trans-regulatory elements(protein)

Transcription

PromoterBasal


Enhancer Region

Activators

Silencer Region

Repressors

Structure of transcription factors

• All transcription factors have more than one DNA

binding domain, matching into the major groove of

DNA double helix with special motifs

– Typical motifs of DNA binding domains:

• Helix-turn-helix (= homeodomain)

• Zinc finger (nuclear receptors)

• Leucine zipper

• Helix-loop-helix

Transcription factors – typical motifs of DNA binding domains

Helix-turn-helixhomeodomain

Zinc finger

Leucine zipper Helix-loop-helix

Function of transcription factors by

DNA binding domains

• Helix-turn-helix (= homeodomain): usually

regulates development of animals

• Zinc finger: functions usually as nuclear

receptors (see next slide)

• Leucin zipper: are usually (proto-)onco-genes

are tumorigenic

• Helix-loop-helix: similarly, they are usually

(proto-)onco-genes are tumorigenic

Nuclear receptors

Nuclear receptors bind the appropriate ligand and get

activated

(ligand is usually a hormone,

e.g. steroid or thyroid hormone)

The activated receptor enters the nucleus

(internalisation)

In the nucleus, receptor acts as transcription factor

(with its zinc-finger motif) impact on transcription

http://www.angelfire.com/sc3/toxchick/endocrinology/endocrinology04.html

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