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Molecular Biology (3/30~4/25, 2007). What is transcription ? How transcription works ? Stages Machinery Molecular mechanism How transcription is regulated ? Regulators Mechanisms Examples of transcriptional regulation Phage strategy RNA silencing. Ch. 9. Ch. 10,11.

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What is transcription ? How transcription works ? Stages Machinery Molecular mechanism

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What is transcription how transcription works stages machinery molecular mechanism

Molecular Biology (3/30~4/25, 2007)

What is transcription?

How transcription works?

Stages

Machinery

Molecular mechanism

How transcription is regulated?

Regulators

Mechanisms

Examples of transcriptional regulation

Phage strategy

RNA silencing

Ch. 9

Ch. 10,11

Ch. 12, 11

沈湯龍 (Tang-Long Shen) 助理教授細胞生物學一號館315室Tel: 3366-4998; E-mail: [email protected]


What is transcription how transcription works stages machinery molecular mechanism

Transcription in Prokaryotes vs. Eukaryotes

Eukaryotic Cell

Prokaryotic Cell

Because there is no nucleus to separate the processes of transcription and translation, when bacterial genes are transcribed, their transcripts can immediately be translated.

Transcription and translation are spatially and temporally separated in eukaryotic cells; that is, transcription occurs in the nucleus to produce a pre-mRNA molecule. The pre-mRNA is typically processed to produce the mature mRNA, which exits the nucleus and is translated in the cytoplasm.


What is transcription how transcription works stages machinery molecular mechanism

Transcription in prokaryotes


The basis of life

The basis of life


What is transcription how transcription works stages machinery molecular mechanism

What is transcription?


What is transcription how transcription works stages machinery molecular mechanism

Central Dogma of Biology:

DNA → RNA → protein

☆ Gene Expression: Transcription

Transcription = DNA → RNA

☆Gene functions (majority) are expressed as the proteins they encode: Translation

Translation = RNA → protein


What is transcription how transcription works stages machinery molecular mechanism

RNA is structurally similar to DNA

But……..


What is transcription how transcription works stages machinery molecular mechanism

Gene Transcription: DNA → RNA

genetic information flows from DNA to RNA

by RNA polymerase

RNA is identical in sequence with one strand of the DNA (but T→U), called coding strand.

  • Four stages of transcription:

  • Promoter recognition and initial melting

  • (binary complex formation)

  • 2. Initiation (ternary complex formation)

  • 3. Elongation

  • 4. Termination


What is transcription how transcription works stages machinery molecular mechanism

Transcription Unit

RNA polymerase

Transcription

unit

binding

release

May include more than one gene

A transcription unitis the distance between sites of initiation and termination by RNA polymerase; may include more than one gene (particularly in prokaryotes).

,

3’

5’

(Primary transcript)

no number 0

mRNA

3’

5’

A relative location

on a linear sequence


What is transcription how transcription works stages machinery molecular mechanism

How transcription works?


Basic principles of transcription

Basic principles of transcription

Template recognition: polymerase and duplex

DNA

Initiation: polymerase* and promoters

Elongation: RNA polymerase

Termination: terminator

abortive

initiation


Initiation

Initiation

  • Binding of an RNA polymerase to the dsDNA

  • (Slide) to find the promoter

  • Unwind the DNA helix

  • Synthesis of the RNA strand at thestart site (initiation site),this position called position +1


What is transcription how transcription works stages machinery molecular mechanism

Transcription Bubble

To fulfill the principle process of transcription, that is

complementary base pairing, a transient bubblehas to be created.

Two strands of DNA are separated

(about 12~14 bp in length).

Template strand is used to synthesize

a complementary sequence of RNA.

The length of RNA-DNA hybrid

within the bubble is about 8~9 bp.

As RNA polymerase moves along the DNA, the transient bubble moves along with and the RNA chain grows continuously.


What is transcription how transcription works stages machinery molecular mechanism

Transcription Bubble

RNA-DNA hybrid length

Ternary Complex:

Polymerase-DNA-RNA

~ 8 to 9 bases, it is short and transient

Function of RNA Polymerase

Unwinding and Rewind DNA

NTPs polymerized to a RNA chain

Moving in the DNA

About 25-base RNA molecule associated with

the ternary complex at any moment.


What is transcription how transcription works stages machinery molecular mechanism

Elongation

Progression of transcription bubble is association with

RNA polymerase movement on DNA

RNA extension

5’

3’

Movement models

  • Sliding:

  • inchworm

DNA rewind behind

DNA unwind ahead

RNA


What is transcription how transcription works stages machinery molecular mechanism

Reaction in Transcription (RNA polymerization)

RNA polymerization

DNA replication

5’ → 3’ ~800 bp/sec

Direction 5’ to 3’

NTP

γ

5

~40 nt/sec

β

4

3

2

Substrates

ATP, UTP, GTP, CTP

Phosphate

α,β,γ

Nucleotide

Ribose

5C -- 1,2,3,4, 5

α

Protein translation

N → C termini ~15 aa/sec

NTP

5

1

4

3

2

NTP

5

γ

3

β

α

NTP

5

3


What is transcription how transcription works stages machinery molecular mechanism

Stages of transcription

(5’)

Promoter

: closed complex

Terminator

(3’)

: open complex

Binary

Promoter clears

Bubble moves on

Ternary

Abortive initiation: to ensure the initiation in a right way.

Movement models

(before the 10th base is added on nascent RNA chain within the bubble)

  • Sliding: common

  • inchworm

Extending RNA chain is accomplished with RNA poly (bubble)

moves along DNA.

The bases after 9th enable added on the growing RNA chain.

move

Recognize termination signal

Release RNA chain (by disrupt RNA:DNA hybrid)

Dissociation of RNA pol


What is transcription how transcription works stages machinery molecular mechanism

Machinery in transcription


What is transcription how transcription works stages machinery molecular mechanism

RNA Pol I

rRNA

RNA Pol II

mRNA

RNA Pol III

tRNA, 5S rRNA

Transcription in Prokaryotes

RNA polymerase

Prokaryotes have a single RNA polymerase

enzyme--synthesizes mRNAs, rRNAs, and tRNAs

Transcribe over > 1000 transcription units. The complexity is modified by

interacting with diverse regulatory factors.

Eukaryotes have three RNA polymerase Enzymes:


E coli rna polymerase

E. coli RNA polymerase

RNA polymerase binds to the promoter

Core enzyme + sigma factor = holoenzyme

155 KD

36.5 KD

11 KD

36.5 KD

70 KD

Initiation only

151 KD

465kD

Both initiation & elongation


What is transcription how transcription works stages machinery molecular mechanism

2 a subunits

Enzyme assembly, Promoter recognition, factor binding

b subunit

Catalytic Center

b' subunit

Catalytic Center

Template-binding

s subunit

Promoter specificity

Structure and functions of E. coli RNA Polymerase


What is transcription how transcription works stages machinery molecular mechanism

Eubacteria RNA polymerase (Pol)

About 7000 RNA polymerase molecules are

present in an E. coli cell.

Most of them are engaged in transcription.

In a short period of time, 2000-5000 Pol molecules

can be synthesized.


E coli polymerase subunit

E. coli Polymerase:α subunit

  • Two identical subunits in the core enzyme

  • Encoded by the rpoA gene

  • Required for core protein assembly

  • May play a role in promoter recognitionandregulatory factors interaction

  • ADP-ribosylation on an arginine upon T4 infection


What is transcription how transcription works stages machinery molecular mechanism

E. coli polymerase: b subunit

  • Encoded by rpoB gene.

  • The catalytic center of the RNA polymerase

  • Rifampicin(used for anti-tuberculosis): bind to the β subunit (12A away from active site), and inhibit transcription initiation. Blocking the path for extending RNA chain beyond 2-3 nts. Mutation in rpoB gene can result in rifampicin resistance.

  • Streptolydigins:resistant mutations are mapped to rpoB gene as well. Inhibits transcription elongation but not initiation.

    3.b subunit may contain two domains responsible for transcription initiation and elongation


What is transcription how transcription works stages machinery molecular mechanism

E. coli polymerase: b’ subunit

  • Encoded by the rpoC gene .

  • Binds two Zn 2+/Mg 2+ ions and may participate in the catalytic function of the polymerase

  • Heparin:binds to the b’ subunit and inhibits transcription in vitro due to it competes with DNA for binding to the polymerase.

    3.b’ subunit may be responsible for binding to the template DNA .


What is transcription how transcription works stages machinery molecular mechanism

E. coli polymerase: s factor

  • Many prokaryotes contain multiple s factors to recognize different promoters. The most common s factor in E. coli is s70. (differential specificity)

  • Binding of the s factor converts the core RNA pol into the holoenzyme.

  • s factor is critical in promoter recognition, by decreasing the affinity of the core enzyme for non-specific DNA sites (104) and increasing the affinity for the corresponding promoter

  • s factor is released from the RNA pol after initiation (RNA chain is 8-9 nt)

  • Less amount of s factor is required in cells than that of the other subunits of the RNA pol.


What is transcription how transcription works stages machinery molecular mechanism

Holoenzyme on promoter recognition

(Core enzyme + sigma factor = holoenzyme)

Core enzyme has the ability to synthesize RNA on a DNA template,

but cannot initiate transcription at the proper sites.

Holoenzyme has ~104-fold lower affinity for loose binding

complexes than core. About 60 min half-life reduce to <1 sec.

Holoenzyme has ~103-fold higher affinity for specific binding

to promoters than core with a half life of several hours.

Totally, sigma factor can result in

107 increase in DNA binding specificity.

Core enzyme does not distinguish between

promoters and other sequences of DNA.


What is transcription how transcription works stages machinery molecular mechanism

Sigma factor is required only for initiation

reversible

Wide range

Faster

Tight binding

Fastest

Less than 10 bases

Slow

Beyond 10 bases leads to elongation


What is transcription how transcription works stages machinery molecular mechanism

Recycle of sigma factor for the utilization of core enzyme

Sigma factor is much less in number than core enzyme

Evidence:

1/3 of sigma factors are not associated with

core enzyme while elongation

recycled

Immediately after initiation


What is transcription how transcription works stages machinery molecular mechanism

Molecular structure of RNA polymerases

in functioning


What is transcription how transcription works stages machinery molecular mechanism

Architecture of RNA polymerases (prokaryotes)

(<100 kD)

Bacterial RNA polymerase (465kD)

T7 RNA polymerase

Multiple subunits:

2α+β+β’+(σ)

25A wide

Enzyme movement

~40nts/sec

~200 nts/sec

Specificity recognition between enzyme and

DNA bases (upstream of startpoint +1)

A channel/groove on the surface ~25A wide

forms a path for DNA.

Path holds for 16 bp in prokaryotes

25 bp in eukaryotes

More DNA bp can reside on the enzyme

Further crystal structure will provide more direct and detailed view in a molecular level.


What is transcription how transcription works stages machinery molecular mechanism

Architecture of RNA polymerases (eukaryotes)

Yeast RNA polymerase contains 12 subunits (10 are shown here)

Nevertheless, it shares similar organization as bacterial one.

A channel/groove on the surface

forms a path for DNA.

Cleft between two

large subunits forms

as an active center

25 bp DNA can be held in the path.


What is transcription how transcription works stages machinery molecular mechanism

Ternary Complex

Channel within RNA polymerase

Active center

+

Enzyme

movement


What is transcription how transcription works stages machinery molecular mechanism

DNA in and out

DNA out

rudder

RNA dissociated

RNA flipped out

Flexible ss DNA

DNA turns

Rigid straight duplex DNA

DNA in

entry

(control by bridge protein)

How many bp(s) in the bubble?


What is transcription how transcription works stages machinery molecular mechanism

Contact among the ternary structure in the active site

These contacts can stabilize the single strand nucleic acid chains.


What is transcription how transcription works stages machinery molecular mechanism

Cycle of making and breaking bonds between enzyme and nucleic acids

nt enters, adds,

and interacts with

the bridge protein

straight

nt still interacts with the

bridge protein, which

leads the protein to

bending due to Pol

moves one bp forward.

bent

Meanwhile, bridge blocks

free nt enters.

straight

Finally, bridge releases

Its interaction with newly

added nt on RNA chain.

Change in conformation of “bridge” protein is closely related

to translocation of the enzyme along the nucleic acid.


What is transcription how transcription works stages machinery molecular mechanism

How does RNA polymerase find promoter sequences?

Directed walk

vs.

Random walk

Random diffusion

(Direct displacement)

No DNA protein is known to work in this way

RNA polymerase found promoters is very faster.

Diffusion in the whole genome cannot support

this fast.

Enzyme moves preferentially from a weak site to a strong site


What is transcription how transcription works stages machinery molecular mechanism

Transitions in shape and size of RNA polymerase during transcription

Covered DNA length

75-80 bp

(-55 to +20)

60 bp

(-35 to +20s)

30-40 bp

(interact w/ RNA pol)


What is transcription how transcription works stages machinery molecular mechanism

How to resume the stalled/pausing RNA polymerase?

Cleavage 3’ end of RNA chain

Backtracks of RNA polymerase as a whole

(Create a 3’-OH for further polymerization)

A constant distance between active site and frond end

To correct mispositioned template during stall

Accessory factors are needed such as:

GreA and GreB for E. coli RNA polymerase

TFIIS for eukaryotic RNA polymerase II

One more function of RNA polymerase:

* cleavage activity is from RNA polymerase itself.

unwind

Rewind

DNA/RNA binding

polymerize RNA


What is transcription how transcription works stages machinery molecular mechanism

Sequence elements in Transcription

Promoter

Coding sequence

Terminator


What is transcription how transcription works stages machinery molecular mechanism

What is a promoter?

  • The sequence of DNA needed for RNA polymerase to bind to the template and accomplish the initiation reaction.

  • Its structure (not transcribed) is the signal (others are needed to be converted into RNAs or proteins).

  • It is a cis-actingsite.

  • Different from sequences whose role is to be transcribed or translated.

What signal (structure) of a promoter provides?


What is transcription how transcription works stages machinery molecular mechanism

AT has only 2 H-bonds, which is easier to be broken

(Open binary complex formation)

(recognition domain

(Closed binary complex formation)

(i.e. the distance of separation between -10 and -35;

intermediate sequence is irrelevant)

Pribnow, D.: Nucleotide Sequence of an RNA Polymerase Binding Site at an Early T7 Promoter. PNAS 72, 784 (1975).

Pribnow, D.: Bacteriophage T7 early promoters: nucleotide sequences of two RNA polymerase binding sites. J. Mol. Biol. 99, 419 (1975).

Schaller, H. et al.: Nucleotide Sequence of an RNA Polymerase Binding Site from the DNA of Bacteriophage fd. PNAS 72, 737 (1975).


What is transcription how transcription works stages machinery molecular mechanism

The sequence comparison of five E. coli promoters

TTGACA

TATAAT

Consensus

Consensus:

the most common base sequence to appear at such points on the DNA helix;

there may be variationsin various organisms


What is transcription how transcription works stages machinery molecular mechanism

Prokaryotic promoters display four conserved features:

1. Startpoint: >90% PURINE (A or G)

2. -10 consensus sequence (Pribnow box)--TAtAaT

T80 A95 t45 A60 a50 T96

3. -35 consensus sequence--TTGACa

T82 T84 G78 A65 C54 a45

4. Distance (spacing) between the -10 and -35 sequences

(The distance is critical in holding the two sites at the appropriate separation for

the geometry of RNA polymerase.)

5. UP element. TA rich sequence upstream of promoter.


What is transcription how transcription works stages machinery molecular mechanism

Functions of promoter domains

-35 recognition domain

Closed binary complex formation

-10 unwinding domain: due to A-T pairs

need lower energy to disrupt (melt)

Open binary complex formation

Sequence around the startpoint (+1 to +30):

influences the initiation event.

Rate of promoter clearance

Other ancillary proteins may help RNA polymerase to recognize deficient promoters.


What is transcription how transcription works stages machinery molecular mechanism

Other structures may exist in a promoter

A-T rich sequence

It interacts with the α subunit of the RNA polymerase,

which to ensure the higher gene expression.

100-fold

variation

in vitro

Down mutation: mutations are tend to be concentrated in the most highly conserved positions.

Up mutation: less cases happen within promoters


What is transcription how transcription works stages machinery molecular mechanism

RNA polymerase-promoter interactions

A promoter with consensus sequences for the -10 and -35 regions (boxed) is shown; the sequences of actual promoters deviate from those shown here.

The "jaws" of RNA polymerase are shown on the right of the molecule. This region of the RNA polymerase would grasp the DNA downstream of the catalytic site. Contacts between RNA polymerase and promoter DNA are shown by the solid lines. Not all contacts occur in every RNA polymerase-promoter interaction, but in all known cases (including promoters activated by regulator proteins), at a minimum, some contacts between  and the 10 region appear to be required.

J Bacteriol, June 1998, p. 3019-3025, Vol. 180, No. 12


10 sequence pribnow box

-10 sequence (Pribnow box)

  • 6 bp sequence which is centered at around the –10 position (Pribnow, 1975).

  • A consensus sequence of TATAAT

  • The first two bases(TA) and the final T are most highly conserved among other E. coli promoters

  • This hexamer is separated by 5 to 9 bp from position +1, and the distance is critical

  • DNA unwinding is initiated at promoter by the polymerase


35 sequence enhances recognition and interaction with the polymerase s factor

-35 sequence: enhances recognition and interaction with the polymerase s factor

  • A conserved hexamer sequence around position –35

  • A consensus sequence of TTGACA

  • The first three positions (TTG) are the most conserved among E. coli promoters.

  • Separated by 16-18 bp from the –10 box in 90% of all promoters


Transcription start site

Transcription startsite

The sequence around the start site influences initiation

  • A purine(A or G) in 90% of all genes

  • Often, there are C and T bases on either side of the start site nucleotide (i.e. CGT or CAT)


Promoter efficiency 1

Promoter efficiency (1)

  • There is considerable variation in sequence between different promoters, and the transcription efficiency can vary by up to 1000-fold .

  • The –35 sequence, -10 sequence, and sequence around the start sites all influence initiation efficiency.


Promoter efficiency 2

Promoter efficiency (2)

  • The sequence of the first 30 bases to be transcribed controls the rate at which the RNA polymerase clears the promoter, hence influences the rate of the transcription and the overall promoter strength .

  • Strand separation in the initiation reaction

  • Some promoter sequence are not strong enough to initiate transcription under normal condition, activating factor is required for initiation. For example, Lac promoter Plac requires cAMP receptor protein (CRP )


Dna unwinding

DNA unwinding

  • Necessary to unwind the DNA so that the antisense strand to become accessible for base pairing, carried out by the polymerase.

  • Negative supercoiling enhances the transcription of many genes but not all (e.g. gyrase) by facilitating unwinding .

  • The initial unwinding of the DNA results in formation of an open complex with the polymerase and this process is referred to as tight binding


What is transcription how transcription works stages machinery molecular mechanism

Supercoiling during transcription

At initiation

∵ Supercoiled structure requires less free energy for the initial melting of DNA

∴ it enhances the efficiency of transcription in vitro

After initiation

DNA is rotated during RNA pol movement; front is overwound and behind is released.

A twin domain on transcribing DNA formed


What is transcription how transcription works stages machinery molecular mechanism

RNA polymerase binds to one face of DNA

(-9 to +3 for unwinding)

Touch down

recognition

contact

extension

initiation


What is transcription how transcription works stages machinery molecular mechanism

Sigma factor controls promoter recognition

Different sigma is used for distinct responses

"housekeeping"

starvation/stationary

extracytoplasmic stress

nitrogen-limitation

flagellar sigma factor

The specificity is determined by recognizing different

consensus sequences in promoters


What is transcription how transcription works stages machinery molecular mechanism

Sigma factors may be organized into cascades

A new sigma factor displaces the previous sigma factor


What is transcription how transcription works stages machinery molecular mechanism

Sigma factors directly contact DNA

which contributes the binding specificities of sigma factors

(most conserved)

melting

2.3

2.4

Release N-terminal

autoinhibition due

conformation change

via interaction with

RNA polymerase

4.2

domain1

conformation change

Coding stand

15A

20A

Free Holo: inside the active site

Complex: displace from active site


What is transcription how transcription works stages machinery molecular mechanism

Transcription initiation

DNA-dependent RNA polymerases are promoter binding,

DNA strand melting,

RNA chain initiation and

nascent RNA chain formation, and

finally escape from the promoter sequences.

abortive RNA synthesis occurs

rate-limiting for the synthesis of productive RNAs

What is the role of sigma factor in abortive initiation/promoter clearance (escape)?


What is transcription how transcription works stages machinery molecular mechanism

Elongation


Elongation

Elongation

  • Add ribonucleotides to the 3’-end (OH group)

  • The RNA polymerase extend the growing RNA chain in the direction of 5’ 3’ (E. coli: 40 nt/sec)

  • The enzyme itself moves in 3’ to 5’ along the antisense DNA strand.


Rna chain elongation

RNA chain elongation

  • σFactor is released to form a ternary complex of the pol-DNA-RNA (newly synthesized), causing the polymerase to progress along the DNA (promoter clearance)

  • Transcription bubble (unwound DNA region, ~ 17 bp) moves along the DNA with RNA polymerase which unwinds DNA at the front and rewinds it at the rear

  • 3’ part of RNA forms hybrid helix (ca. 12bp) with antisense DNA strand.

  • The E. coli polymerase moves at an average rate of ~ 40 nt per sec, depending on the local DNA sequence.


Termination

Termination

  • The dissociation of the transcription complex from the template strand and separation of RNA strand from DNA

  • Occurring at the terminator (often stem-loop or hairpin structure), some need rho protein as accessory factor.

it is a regulatory event

Hence, it is possible to readthrough the terminator (anti-termination)

in a signal-dependent manner.


What is transcription how transcription works stages machinery molecular mechanism

RNA chain termination

  • Termination: dissociation of RNA > re-annealing of DNA > release of RNA pol

  • Terminator sequence (stop signal):

    • RNA hairpin very common

    • Accessory rho protein

The DNA sequences required for termination are

located prior to the terminator sequence.

Formation of a hairpin in the RNA may be necessary


What is transcription how transcription works stages machinery molecular mechanism

RNA hairpin structure: an intrinsic terminator

near the base of the stem.

Hairpin leads RNA pol to slow/pause

The rU.dA RNA –DNA hybrid has an unusually weak base-paired structure;

it requires the least energy of any RNA-DNA hybrid to break the association

between the two strands.


What is transcription how transcription works stages machinery molecular mechanism

A model for intrinsic termination


Rho dependant termination

Rho-dependant termination

  • Some genes contain terminator sequences requiring an accessory factor,the rho protein (ρ) to mediated transcription termination

  • Rho binds to specific sites in the single-stranded RNA

  • Rho hydrolyses ATP and moves along the nascent RNA towards the transcription complex then enables the polymerase to terminate transcription


What is transcription how transcription works stages machinery molecular mechanism

Termination efficiency determinants:

@ The Sequence of the hairpin

@ The length of the U-run

@ Sequences both upstream and downstream of the intrinsic terminator

@ Ancillary proteins

@ others

rich

poor

A bias sequence preceding actual terminator site (RNA) is important for termination efficiency

(rho dependent terminator).


What is transcription how transcription works stages machinery molecular mechanism

~275kD


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