transcription in prokaryotes-akw

8/10/2019 Transcription in Prokaryotes-AKW

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Agustin Krisna Wardani

Transcription in Prokaryotes


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Gene Express ion

1. Transcriptioncopies the information of a DNA

sequence (the gene) into corresponding

information in an RNA sequence.

2. Translationconverts this RNA sequence intothe amino acid sequence of a polypeptide.

The expression of a gene to form a polypeptide

occurs in two major steps:


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The central dogma of molecular biology


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The synthesis of RNA molecules using

DNA strands as the templates so that the

genetic information can be transferred

from DNA to RNA.

Transcription


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DNA vs RNA

RNA much shorterthan DNA

-DNA contains the code for making lots of different

proteins.

-Messenger RNA contains the information to make justone protein

DNA has two strands arranged in a double helix. RNA

consists of a single strand.

DNA (deoxyribonucleic acid) has a backbone of alternating

deoxyriboseand phosphate groups. In RNA (ribonucleicacid), the sugar ribosereplaces deoxyribose.

RNA uses the base uracil (U) rather than thymine (T):

A---U, G---C


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RNA (ribonucleic acid) has the sugar ribose

replaces deoxyribose.

RNA (RiboNucleic Acid)

DNA (DeoxyriboNucleic Acid)


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RNA uses the base uracil (U) rather than thymine (T):

The only difference between the two

molecules is the presence or absence of

the CH3group.


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Similarity between

replication and transcription

Both processes use DNA as the

template.

Phosphodiester bondsare formed in

both cases.

Both synthesis directionsare from5to 3.


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replication transcription

template double strands single strand

primer yes no

Enzyme DNA polymerase RNA polymerase

product dsDNA ssRNA

base pairA-T, G-C

The Amazing Gene Code (TAGC).A-U, T-A, G-C

Differences between

replication and transcription


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Transcriptional Control

DNA

RNA

protein

Environmental change

Turn gene(s) on/off

Proteins to deal withnew environment

Very important to:

1. express genes when needed

2. repress genes when not needed


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The wholegenomeof DNA needs to be

replicated, but only small portion of genome

is transcribedin response to the

development requirement, physiological

need and environmental changes.

DNA regions that can be transcribed into

RNA are called structural genes.


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DNA

RNA

protein

TranscriptionInitiation

Elongation

Termination

ProcessingCapping

Splicing

Polyadenylation

Turnover

Translation

Protein processing

Many places for control


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Prokaryotic Transcription

Operons

Groups of related genes transcribed

by the same promoter

Polycistronic RNA

(one mRNA code several genes)

Multiple genes transcribed

as ONE TRANSCRIPT

No nucleus, so transcription and

translation can occur simultaneously


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Each transcriptable region is called

operon.

One operon includes several structural

genesand upstream regulatorysequences(or regulatory regions).

The promoteris the DNA sequence that

RNA-pol can bind. It is the key pointfor the transcription control.

Recognition of Origins


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Nisin Operon


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Template and Enzymes


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Template

The template strandis the strandfrom which the RNA is actuallytranscribed. It is also termed as

antisensestrand.

The coding strandis the strandwhose base sequence specifies the

amino acid sequence of the encodedprotein. Therefore, it is also called assensestrand.


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G C A G T A C A T G T C5' 3'

3' C G T C A T G T A C A G 5' templatestrand

codingstrand

transcription

RNAG C A G U A C A U G U C5' 3'


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RNA Polymerase

The enzyme responsible for the RNA

synthesis is DNA-dependent RNA

polymerase.

The prokaryotic RNA polymeraseis a

multiple-subunit protein of ~480kD.

Eukaryotic systemshave three kinds of

RNA polymerases I, II, III), each of which

is a multiple-subunit protein and

responsible for transcription of different

RNAs.


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Types of RNA

Messenger RNA (mRNA) genes that encodeproteins

Ribosomal RNA (rRNA) form the core ofribosomes

Transfer RNA (tRNA) adaptors that link

amino acids to mRNA during translation


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Elongation

Termination

ProcessingCapping

Splicing

Polyadenylation

Turnover

Translation

Protein processing

Control of initiation

usually most

important.


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Three phases: initiation, elongation,

and termination.

The prokaryotic RNA-polcan bind to

the DNA template directlyin the

transcription process.

The eukaryotic RNA-polrequires co-factorsto bind to the DNA template

together in the transcription process.

General concepts


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Initiation phase: RNA-pol recognizesthe promoter and starts thetranscription.

Elongation phase: the RNA strand iscontinuously growing.

Termination phase: the RNA-pol stopssynthesis and the nascent RNA isseparated from the DNA template.

Transcription of Prokaryotes


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a. Initiation

RNA-pol recognizesthe TTGACAregion, and slidesto the TATAATregion, then opensthe DNA duplex.

The unwound region is about 171 bp.


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Initiation

RNA polymerase

Promoter DNA

RNAP binding sites


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RNA Polymerase in prokaryote


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Initiation

RNA polymerase

4 core subunits

Sigma factor ()

determines promoter

specificity

Core + = holoenzyme

Binds promoter sequence

Catalyzes open complex and

transcription of DNA to RNA


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RNA Polymerase II in eukaryote


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Promoter


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The first thing that the enzyme has to do is to find the

start of the gene on the coding strand of the DNA.

These base sequences are known as promoter

sequences.


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5'

3'

3'

5'

regulatorysequences

structural gene

promotorRNA-pol

Promoter


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5'

3'

3'

5'-50 -40 -30 -20 -10 1 10

start -10region

T A T A A TA T A T T A

(Pribnow box)

-35

region

T T G A C AA A C T G T

Prokaryotic promoter

Consensus sequence


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Consensus Sequence


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The -35 region of TTGACAsequence

is the recognition siteand the

binding site of RNA-pol.

The -10 region of TATAATis the

region at which a stablecomplex of

DNA and RNA-pol is formed.

RNAP binds specific promoter


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RNAP binds specific promoter

sequences

Sigma factors recognize consensus

-10 and -35 sequences


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RNA polymerase promoters

TTGACA TATAAT

Deviation from consensus -10 , -35 sequence leads to

weaker gene expression


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Bacterial sigma factors

Sigma factors are transcription factors

Different sigma factors bind RNAP and recognize

specific -10 ,-35 sequences

Helps melt DNA to expose transcriptional start site Promote broad changes in gene expression

E. coli7 sigma factors

B. subtilis18 sigma factors

Generally, bacteria that live in more variedenvironments have more sigma factors


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Sigma factors

E. colican choose between 7 sigma factors and about 350

transcription factors to fine tune its transcriptional output

An Rev Micro Vol. 57: 441-466T. M. Gruber

Sigma subunit Type of gene controlled # of genes controlled

RpoD Growth/housekeeping ~1000

RpoN N2; stress response ~15

RpoS Stationary phase, virulence ~100

RpoH Heat shock ~40

RpoF Flagella-chemotaxis ~40

RpoE ? ~5

FecI Ferric citrate transport ~5

Extreme heat shock, unfolded proteins

70

54

S

S

F

32


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elongation

RNA-pol, DNA form a complex called

the transcription bubble.

The 3segmentof the nascent RNA

hybridizes with the DNA template, and

its 5endextends out thetranscription bubble as the synthesis

is processing.


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Once the enzyme has attached to the DNA, itunwinds the double helix over a short length, and

splits the two strands apart. This gives a "bubble" in

which the coding strand and template strand are

separated over the length of about 10 bases.


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Transcription bubble


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the enzyme moves along the DNA, zipping it up

again behind it. Essentially it moves the bubble

along the chain, adding new nucleotides all thetime. The growing RNA tail becomes detached from

the template strand as the enzyme moves along.

RNA s nthesis


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RNA synthesis


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Elongation

Termination

ProcessingCapping

Splicing

Polyadenylation

Turnover

Translation

Protein processing


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How does the enzyme know where to stop after it

reaches the end of the gene?

there will be a term inat ion sequenceof bases.

Once the enzyme gets to those, it stops adding new

nucleotides to the chain and detaches the RNA

molecule completely from the template chain.


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Transcriptional Termination

Bacteria need to end transcription at the

end of the gene

2 principle mechanisms of termination in

bacteria:

Rho-independent (more common)

Rho-dependent


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Rho-independent termination

Termination sequence has 2 features:

Series of U residues

GC-rich self-complimenting region

GC-rich sequences bind forming stem-loop

Stem-loop causes RNAP to pause

U residues unstable, permit release of RNA chain


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Rho-dependent termination

Rho is protein (50kDa) that terminatetranscription

Rho attach rut (rho utilization site),Binds the 70-80 base segment of RNA

Rho moves along RNA and catch up toRNAP, then unwinds DNA/RNA hybrid

Rho has ATPase activity which can

induce release of the polymerase fromDNA.


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RNA hairpins followed by a run of U residues in rho independent

terminator, but not in rho dependent terminator of transcription


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Quorum Sensing

Bacteria produce and secrete chemical signal

molecules (autoinducers)

Concentration of molecules increases with

increasing bacterial density When critical threshold concentration of

molecule is reached, bacteria alter gene

expression

Way for communities of bacteria to talk to

each other


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transcription in prokaryotes-akw

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