TRANSCRIPTION IN PROKARYOTES

• Basic principles of transcription

• Escherichia coli RNA polymerase

• The E. coli s70 promoter

• Transcription in prokaryotes

DNA

RNA

PROTEIN

today’s focus

Transcription of RNA (rRNA, tRNA, mRNA)

• RNA transcribed from coding strand of DNA

• RNA single stranded, may fold back on self and partially pair

• RNA carries uracil, while DNA carries thymine

• mRNA carries message to be translated into a polypeptide product

• tRNA and rRNA function at the RNA level

Potential Regulated Steps

transcription

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export

translation

cap AAAAAAAAAA

cap AAAAAAAAAA

folding

transcriptional translational coupling (why wait?)

• Many Pols simultaneously transcribing the gene

• Ribosomes simultaneously translating protein

• Does not occur in eukaryotes

gene 1 gene 2

DNA

TranscriptionThe synthesis of a single-stranded RNA from a

double-stranded DNA template. RNA synthesis occurs in the 5’3’direction and its sequence corresponds to that of the DNA strand which is known as the sense strand.

The template of RNA synthesis is the antisense strand.

Necessary components: promoter/template, RNA polymerase, rNTPs, terminator/template

(-) strand is antisense strand. (+) strand is sense strand

5’-CGCTATAGCGTTT-3’ DNA nontemplate (+) strand 3’-GCGATATCGCAAA-5’ DNA template (-) strand

5’-CGCUAUAGCGUUU-3’ RNA transcript

Basic principles of transcription

Initiation: polymerase and promoters

Elongation: polymerase

Termination: terminator

Initiation:RNA polymerase is the enzyme responsible for transcription. It binds to specific DNA sequences called promoters to initiate RNA synthesis. These sequences are upstream (to the 5’ end) of the region that codes for protein, and they contain short, conserved DNA sequences which are common to different promoters. The RNA polymerase binds to the dsDNA at a promoter sequence, resulting in local DNA unwinding. The position of the first synthesized base of the RNA is called the start site and is designate as position +1

Initiation • Binding of an RNA polymerase to the

dsDNA

• Slide to find the promoter

• Unwind the DNA helix

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

Promoter+1

Elongation: RNA polymerase moves along the DNA and sequentially synthesizes the RNA chain. DNA is unwound ahead of the moving polymerase, and the helix is reformed behind it.

Elongation

• Add ribonucleotides to the 3’-end

• 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.

•The dissociation of the transcription complex from the template strand

•Occurring at the terminator

Termination: RNA polymerase recognizes the terminator which causes no further ribonucleotides to be incorporated. This sequence is commonly a hairpin structure. Some terminators require an accessory factor called rho for terminaton

Termination site for RNA polymerase in bacteriaTermination site for RNA polymerase in bacteria

• Direct termination: The RNA hairpin loop of GC sequences and section of U residues appear to serve as signal for RNA polymerase release and termination of transcription.

•Alternative termination method: rho protein binds to specific sequences referred to as rut. rho pulls RNA polymerase off RNA.

DNA helix is transiently unwound within the polymerase while mRNA is synthesized

polymerase moves downstream

As it synthesizes mRNA

Note: unlike helicase, ATP not required

Transcription

2. Requires DNA for activity and is most active with a double-stranded DNA as template. 5’ 3’ synthesis

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

RNA polymerase: synthesis of RNA strand from DNA template.

1. Requires no primer for polymerization

3. Require Mg2+ for RNA synthesis activity

4. All RNA polymerases lack 3’ 5’ exonuclease activity, and one error usually occurs when 104 to 105 nucleotides are incorporated.

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.

5. usually are multisubunit enzyme, but not always.

7. E. coli has a single DNA-directed RNA polymerase that synthesizes all types of RNA.

6. Different from organism to organism

E.Coli RNA polymeraseRNA polymerase is responsible for RNA synthesis (Transcription). The core enzyme, consisting of 2, 1 , 1 ’,and 1 subunits, is responsible for transcription elongation. The sigma factor (s), is also required for correct transcription initiation. The complete enzyme, consisting of the core enzymes plus the factor s, is called the holoenzyme.

E. coli RNA polymerase

E. coli RNA polymerase

Both initiation & elongation

Initiation only

36.5 KD

36.5 KD

151 KD

155 KD

11 KD

70 KD

• RNA synthesis rate: 40 nt per second at 37oC

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

RNA polymeraseRNA polymerase

E. coli polymerase: subunit

• Two identical subunits in the core enzyme

• Required for core protein assembly ,but has no clear function

• May play a role in promoter recognition. Once the subunit modified, it reduces the binding affinity to promoter region.

1. The catalytic center of the RNA polymerase

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

E. coli polymerase: subunit

E. coli polymerase: s factor

Sigma factor is a separate component from the core enzyme. E. coli encodes several s factors, the most common being s70. A s factor is required for initiation at the correct promoter site. It does this by decreasing binding of the core enzyme to nonspecific DNA sequences and increasing specific promoter binding. The s factor is released from the core enzyme when the transcript reaches 8-9 nt in length.

1. Many prokaryotes contain multiple s factors to recognize different promoters. The most common s factor in E. coli is s70.

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

3. 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

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

5. Less amount of s factor is required in cells than that of the other subunits of the RNA pol.(30% present)

E. coli polymerase: s factor

The E coli σ70 promoter

1. Promoter sequences:

-10 sequence and -35 sequence

2. Transcription Start site

3. Promoter efficiency

Promoters: contain conserved sequences which are required for specific binding of RNA pol

and transcription initiation

ATACGTATGC

+1promoter terminatorTranscribed region

RNA

DNA

Transcription

Antisense strand

Roles in transcription: different promoters result in differing efficiencies of transcription initiation, which in turn regulate transcription.

Promoter sequence

• Lies upstream of the start site of transcription ( position +1),thus the promoter sequences are assigned a negative number

• Contains short conserved sequences critical for specific binding of RNA polymerase and transcription initiation

s70 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

• up to position –40: critical for promoter function

• -10 and –35 sequence:particularly important for promoter function

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

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

+1-35 sequence -10 sequence

-10 sequence (Pribonow box)• 6 bp sequence which is centered at around the –

10 position

• 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 between 5 and 8 bp from position +1,and the distance is critical

-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

• Is a purine in 90% of all genes

• G is more common at position +1 than A

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

The sequence around the start site influences initiation

The sequences of five E. coli promoters

E. coli promoter sequences for 13 genes

• Promoter sequences for mRNAs. RNA polymerase seeks out the consensus sequences for proper orientation for binding to initiate transcription.

• Note promoter sites have regions of similar sequences at the -35 region and -10 region.

• Minus numbers represent bases upstream of mRNA start point, +1 is the first base in the RNA transcript.

Promoter structure in prokaryotes

5’ PuPuPuPuPuPuPuPu AUG

Promoter

+1 +20-7-12-31-36

5’mRNA

mRNA

TTGACAAACTGT

-35 region

TATAATATATTA

-10 region

84 79 53 45%82T T G

64AC A

79T

44T

96%T

95A

59A

51A

consensus sequences

-30 -10

transcription start site

Pribnow box

+1[ ]

trancription initiation, elongation and termination

Promoter binding

DNA unwinding

RNA chain initiation

RNA chain elongation

RNA chain termination

Rho-dependent termination

Promoter bindingThe σ factor enhances the specificity of the core polymerase for promoter binding. The polymerase finds the promoter –35 and –10 sequences by sliding along the DNA and forming a closed complex with the promoter DNA

Binding

Unwinding

Initiation

Elongation

termination

s

s (sigma subunit) allows RNA polymerase to recognize and bind specifically to promoter regions.

E. coliE. coli RNA polymerase + RNA polymerase + ss subunit subunit

Promoter binding

• The σ factor enhances the specificity of the core α2ββ’ωRNA polymerase for promoter binding

• The polymerase finds the promoter –35 and –10 sequences by sliding along the DNA extremely rapidly and forming a closed complex with the promoter DNA

(The initial complex of the polymerase with the base-paired promoter DNA)

DNA unwinding : around 17 bp of the DNA is unwound by the polymerase, forming an open complex. DNA unwinding at many promoters is enhanced by negative DNA supercoiling. However, the promoters of the genes for DNA gyrase subunits are repressed by negative supercoiling.

DNA unwinding

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

• Negative supercoiling enhances the transcription of many genes but not all 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

RNA chain initiation• No primer is needed • Start with a GTP or ATP • The first 9 nt are incorporated without

polymerase movement along the DNA or σfactor release

• The RNA polymerase goes through multiple abortive chain initiations, which are important for the overall rate of transcription

• The minimum time for promoter clearance is 1-2 seconds (a long event)

RNA chain elongation• σFactor is released to form a ternary complex of the

newly synthesized RNA, 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. 10-12 nucleotides at the 5’-end of the RNA are constantly base-paired with the DNA template strand.

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

RNA chain termination: Self-complementary sequences at the 3’-end of genes cause hairpin often has a high content of G-C base pairs giving it high stability, causing the polymerase to pause. The hairpin is often followed by four or more Us which result in weak RNA-antisense DNA strand binding . This favors dissociation of the RNA strand, causing transcription termination.

Rho-dependent termination: Some genes contain terminator sequences which require an additional protein factor, rho, for efficient transcription termination. Rho binds to specific sites in single-stranded RNA. It hydrolyzes ATP and moves along the RNA towards the transcription complex, where it enables the polymerase to terminate transcription

RNA hairpin

1. RNA transcript is self-complementary

2. GC-rich favoring the base pairing stability and causing the polymerase to pause

3. Followed by a sequence of four or more Us which result in weak RNA-antisense DNA strand binding

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

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

stability3. Rho-dependent and -independent

Termination.

TerminatorA specific DNA sequence where the

transcription complex dissociate.

Class 1: Rho protein (r) independent. Contains (1) self-complementary region that can form a stem-loop or hairpin secondary structure, and (2) a run of adenylates (As) in the template strand that are transcribed into uridylates (Us) at the end of the RNA.

Class 2: Rho protein (r) dependent. Contains only the self-complementary region that can form a stem-loop or hairpin secondary structure.

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

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

2. Rho-dependent Termination.

No U stretch at the 3’ end of the RNA

Rho protein (hexameric protein) binds to certain RNA structure moves along the nascent RNA toward RNA pol complex stop at the rho-dependent transcription terminator.

RNA polymerase/transcription and DNA polymerase/replication

RNA pol DNA pol

Template dsDNA is better

Require primer No

Initiation promoter

elongation 40 nt/ sec

terminator Synthesized RNA

ssDNA

Yes

origin

900 bp/sec

Template DNA

Thanks