Molecular Biology Course Section K: Transcription in prokaryotes

•Molecular Biology Course

Section K: Transcription in prokaryotes

K1 Basic principles of transcriptionK1 Basic principles of transcription

K2 Escherichia coli RNA polymeraseK2 Escherichia coli RNA polymerase

K3 The E. coli 70 promoterK3 The E. coli 70 promoter

K4 transcription process. K4 transcription process.

An overview, the process of RNA synthesis ( initiation, elongation, termination)

Properties, subunit, subunit, ’ subunit, sigma () factor

Promoter, 70 size, -10 sequence, -35 sequence, transcription start site, promoter efficiency

Promoter binding, unwinding, RNA chain initiation, elongation, termination ( factor)

Section K: Transcription in prokaryotes


K1: Basic principles of transcription

1.Transcription: an overview (comparison with replication)

2.The process of RNA synthesis: initiation, elongation, termination


K1-1: Transcription: an overview

Key terms defined in this section (Gene VII)

Coding strand of DNA has the same sequence as mRNA.Downstream identifies sequences proceeding further in the direction of expression; for example, the coding region is downstream of the initiation codon.

Gene X

Primary transcriptm7Gppp AAAAAn

+1

upstream downstream

mRNA

Upstream identifies sequences proceeding in the opposite direction from expression; for example, the bacterial promoter is upstream from the transcription unit, the initiation codon is upstream of the coding region.

Transcription unit is the distance between sites of initiation and termination by RNA polymerase; may include more than one gene.

Promoter is a region of DNA involved in binding of RNA polymerase to initiate transcription

RNA Terminator is a sequence of DNA, represented at the end of the transcript, that causes RNA polymerase to terminate transcription.

RNA polymerases are enzymes that synthesize RNA using a DNA template (formally described as DNA-dependent RNA polymerases).

Primary transcript is the original unmodified RNA product corresponding to a transcription unit.

Replication: synthesis of two DNA molecules using both parental DNA strands as templates. Duplication of a DNA molecule. 1 DNA molecule 2 DNA molecules

Transcription: synthesis of one RNA molecule using one of the two DNA strands as a template.1 DNA molecule 1 RNA molecule


Replication-synthesis of the leading strand

the same direction as the replication fork moves

Review of replication

Replication- Synthesis of the Okazaki fragments

Opposite to the replication fork movement

Review of replication

Coupling the synthesis of leading and lagging strands with a dimeric DNA pol III (E. coli)

TranscriptionK1: Basic principles of transcription

1.RNA synthesis occurs in the 5’3’ direction and its sequence corresponds to the sense strand (coding strand).

2.The template of RNA synthesis is the antisense strand (template strand).

3.Phosphodiester bonds: same as in DNA

4.Necessary components: RNA polymerase, transcription factors, rNTPs, promoter & terminator/template


K1-2: The process of RNA synthesis

1.initiation

2.elongation

3.termination


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Promoter Terminator

Transcribed region Sense strand

Antisense strandDNA

RNATranscription

+1

Fig. 2. Structure of a typical transcription unit

Is transcribed region equal to coding region? Why?


Initiation (template recognition) 1. Binding of an RNA polymerase to the

dsDNA

2. Slide to find the promoter

3. Unwind the DNA helix

4. Synthesis of the RNA strand at the start site (initiation site), this position called position +1

Link


Elongation

• Covalently adds ribonucleotides to the 3’-end of the growing RNA chain.

• The RNA polymerase extend the growing RNA chain in the direction of 5’ 3’

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

Link


Termination

• Ending of RNA synthesis: the dissociation of the RNA polymerase and RNA chain from the template DNA at the terminator site.

• Terminator: often contains self-complementary regions which can form a stem-loop or hairpin structure in the RNA products (see K4 for details)

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K2 Escherichia coli RNA polymerase

1.E. coli RNA polymerase 2. subunit3. subunit4. ’ subunit5.sigma () factor

K2: E. coli RNA polymerase

K2-1 E. coli RNA polymerase

Synthesis of single-stranded RNA from DNA template.

1. Requires no primer for polymerization2. Requires DNA for activity and is most active

with a double-stranded DNA as template.3. 5’ 3’ synthesis4. Require Mg2+ for RNA synthesis activity5. lacks 3’ 5’ exonuclease activity, and the err

or rate of nucleotides incorporation is 10-4 to 10-5. Is this accuracy good enough for gene expression??

6. usually are multisubunit enzyme.

K2: E. coli RNA polymerase RNA polymerase

(NMP)n + NTP (NMP)n+1 + PPi

E. coli polymerase1. E. coli has a single DNA-directed RNA polymer

ase that synthesizes all types of RNA. 2. One of the largest enzyme in the cells3. Consists of at least 5 subunits in the holoenzym

e, 2 alpha (), and 1 of beta (), beta prime (’), omega () and sigma () subunits

4. Shaped as a cylindrical channel that can bind directly to 16 bp of DNA. The whole polymerase binds over 60 bp.

5. RNA synthesis rate: 40 nt per second at 37oC


E. coli RNA polymerase

Both initiation & elongation

Initiation only

36.5 KD

36.5 KD

151 KD

155 KD

11 KD

70 KD


The polymerases of bacteriophage T3 and T7 are smaller single polypeptide chains, they synthesize RNA rapidly (200 nt/sec) and recognize their own promoters which are different from E. coli promoters.

RNA polymerase differs from organism to organism


K2-2: subunit


E. coli polymerase: subunit • Two identical subunits in the core enzyme • Encoded by the rpoA gene • Required for assembly of the core enzyme• Plays a role in promoter recognition. Experim

ent: When phage T4 infects E. coli, the α subunit is modified by ADP-ribosylation of an arginine. The modification is associated with a reduced affinity for the promoters formerly recognized by the holoenzyme.

• plays a role in the interaction of RNA polymerase with some regulatory factors


K2-3&4: and ’ subunit


1. is encoded by rpoB gene, and ’ is encoded by rpoC gene .

2. Make up the catalytic center of the RNA polymerase

3. Their sequences are related to those of the largest subunits of eukaryotic RNA polymerases, suggesting that there are common features to the actions of all RNA polymerases.

4. The subunit can be crosslinked to the template DNA, the product RNA, and the substrate ribonucleotides; mutations in rpoB affect all stages of transcription. Mutations in rpoC show that ’ also is involved at all stages.

subunit may contain two domains responsible for transcription initiation and elongation

•Rifampicin ( 利福平 ) ： has been shown to bind to the β subunit, and inhibit transcription initiation by prokaryotic RNA pol. Mutation in rpoB gene can result in rifampicin resistance.

•Streptolydigins( 利迪链菌素 ) ： resistant mutations are mapped to rpoB gene as well. Inhibits transcription elongation but not initiation.


’ subunit • Binds two Zn 2+ ions and may participate in t

he catalytic function of the polymerase • Hyparin ( 肝素 ) ： binds to the ’ subunit an

d inhibits transcription in vitro. • Hyparin competes with DNA for binding to the

polymerase.2. ’ subunit may be responsible for binding to

the template DNA .


K2-5: Sigma () factor


1. Many prokaryotes contain multiple factors to recognize different promoters. The most common factor in E. coli is 70.

2. Binding of the factor converts the core RNA pol into the holoenzyme.

3. 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

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

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


K3: The E. coli 70 promoterK3: The E. coli 70 promoter1. Promoter2. 70 size3. -10 sequence4. -35 sequence5. transcription start s

ite6. promoter efficiency

K3: The E. coli 70 promoter

K3-1: PromoterK3-1: Promoter1. The specific short conserved DNA sequences:2. upstream from the transcribed sequence, and

thus assigned a negative number (location) 3. required for specific binding of RNA Pol. an

d transcription initiation (function) 4. Were first characterized through mutations th

at enhance or diminish the rate of transcription of gene

Different promoters result in differing efficiencies of transcription initiation, which in turn regulate transcription.

Promoter Terminator

Transcribed region Sense strand

Antisense strandDNA

RNATranscription

+1


K3-2,3&4: 70 promoterK3-2,3&4: 70 promoter


• Consists of a sequence of between 40 and 60 bp• -55 to +20: bound by the polymerase• -20 to +20: tightly associated with the

polymerase and protected from nuclease digestion by DNaseΙ(see the supplemental)

• Up to position –40: critical for promoter function (mutagenesis analysis)

• -10 and –35 sequence: 6 bp each, particularly important for promoter function in E. coli

---5-8 bp--- GC T A

TTGACA TATAAT-----16-18 bp-------

+1-35 sequence -10 sequence


-10 sequence (Pribonow box)1. The most conserved sequence in 70 promoter

s at which DNA unwinding is initiated by RNA Pol.

2. A 6 bp sequence which is centered at around the –10 position, and is found in the promoters of many different E. coli gene.

3. The consensus sequence is TATAAT. The first two bases (TA) and the final T are most highly conserved.

4. This hexamer is separated by between 5 and 8 bp from position +1, and the distance is critical.


-35 sequence: enhances recognition and interaction with the polymerase 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


• Footprinting is a technique derived from principles used in DNA sequencing. It is used to identify the specific DNA sequences that are bound by a particular protein.

RNA Polymerase Leaves Its FootPrint on a Promoter

Supplemental material


Footprinting


Footprinting

K3-5: Transcription start siteK3-5: Transcription start site


• Is a purine in 90% of all gene

• G is more common at position +1 than A

• There are usually a C and T on either side of the start nucleotide (i.e. CGT or CAT)

The sequences of five E. coli promoters


K3-6: promoter efficiencyK3-6: promoter efficiency


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

1. The –35 sequence constitutes a recognition region which enhances recognition and interaction with the polymerase factor.

2. The -10 sequence is important for DNA unwinding.

3. The sequence around the start site influence initiation efficiency.

4. 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.

Some promoter sequence are not sufficiently similar to the consensus sequence to be strongly transcribed under normal condition, thus activating factor is required for efficient initiation.

Example: Lac promoter P lac requires activating protein, cAMP receptor protein (CRP ), to bind to a site on the DNA close to the promoter sequence in order to enhance polymerase binding and transcription initiation.

Weak promoters and activating factor


K4 Transcription process K4 Transcription process 1.Promoter binding2.DNA unwinding3.RNA chain initiation4.RNA chain elongation5.RNA chain termination

( factor)

1.Promoter bindingK4 Transcription process

The searching process is extremely rapidlyThe searching process is extremely rapidly

Closed complex: the initial complex of the polymerase with the base-paired promoter DNA)

Closed complex: the initial complex of the polymerase with the base-paired promoter DNA)

and –10 region

Link

• The RNA polymerase core enyzme, 2’ has a general non-specific affinity for DNA, which is referred to as loose binding that is fairly stable.

• The addition of factor to the core enzyme markedly reduces the holoenzyme affinity for non-specific binding by 20 000-fold, and enhances the holoenzyme binding to correct promoter sites 100 times.

• Overall, factor binding dramatically increases the specificity of the holoenzyme for correct promoter-binding site.

The role of factor in promoter binding K4 Transcription process

K4 Transcription process

2. DNA 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

+1

• It is necessary to unwind the DNA so that the antisense strand to become accessible for base pairing and RNA synthesis.

• Negative supercoiling enhances the transcription of many genes, since it facilitates unwinding. Some promoters are not.

• Exceptional example: promters for the enzyme subunits of DNA gyrase are inhibited by negative supercoiling, serving as an elegant feedback loop for DNA gyrase expression.

Negative supercoiling & unwindingK4 Transcription process

3. RNA chain initiation

+1

The polymerase initially incorporates the first two nucleotides and forms a phosphodiester bond.


Starts with a GTP or ATP

Abortive initiation

• The RNA pol. goes through multiple abortive initiations before a successful initiation, which limits the overall rate of transcription

• The minimum time for promoter clearance is 1-2 seconds (a long event, the synthesis is 40 nt/ sec)

The first 9 nt are incorporated without polymerase movement along the DNA. Afterward, there is a significant probability that the chain will be aborted.


4. RNA chain elongation


• Promoter clearance: when initiation succeeds, the enzyme releases factor and forms a ternary complex of polymerase-DNA-nascent RNA, causing the polymerase to progress along the DNA to allow the re-initiation of transcription.


Transcription bubble:

1. containing ~ 17 bp of unwound DNA region and the 3’-end of the RNA that forms a hybrid helix about 12 bp.

2. moves along the DNA with RNA polymerase which unwinds DNA at the front and rewinds it at the rear.

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


Transcription bubbleTranscription bubble

5. RNA chain termination

1.Termination occurs at terminator DNA sequences.

2.The most common stop signal is an RNA hairpin (self-complement structure)

commonly GC-rich to favor the structure stability

3. Rho-dependent and Rho-independent Termination.

TerminatorA specific DNA sequence where the

transcription complex dissociateRho protein () independent terminator contains:

(1) self-complementary region that is G-C rich and can form a stem-loop or hairpin secondary structure. GC-rich favouring the base pairing stability and causing the polymerase to pause.

(2) a run of adenylates (As) in the template strand that are transcribed into uridylates (Us) at the end of the RNA, resulting in weak RNA-antisense DNA strand binding.

A model for -independent termination of transcription in E. coli.

The A-U base-pairing is less stable that favors the dissociation

1. Contains only the self-complementary region

2. Requires protein for termination

3. protein binds to specific sites in the single-stranded RNA

4. protein hydrolyzes ATP and moves along the nascent RNA towards the transcription complex then enables the polymerase to terminate transcription

Rho protein () dependent terminator

RNA polymerase/transcription and DNA polymerase/replication

RNA pol DNA pol

Template dsDNA is better than ssDNA

dsDNA

Require primer No Yes

Initiation promoter origin

elongation 40 nt/ sec 900 bp/sec

terminator Synthesized RNA Template DNA

K: supplemental 1

In any chromosome, different genes may use different strands as template (Fig. 25-2).

Thanks

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Molecular Biology Course Section K: Transcription in prokaryotes