Lecture #9

Prokaryotic & Eukaryotic Transcription

Transcription is __ to __ on a template that is __ to __

5’ to 3’, 3’ to 5’

coding strand (nontemplate strand)

the DNA strand that has the same sequence as the mRNA and is related by the genetic code to the protein sequence that it represents

RNA Polymerase

An enzyme that synthesizes RNA using a DNA template (formally described as a DNA-dependent RNA polymerase)

RNA sequence is complementary to ________ strand and ________ to coding strand.

template, coding

Promoter

a region of DNA where RNA polymerase binds to initiate transcription

Startpoint

The position on DNA corresponding to the first base incorporated into RNA

Terminator

A sequence of DNA that causes RNA polymerase to terminate transcription

Transcription Unit

The sequence between sites of initiation and terminations by RNA polymerase; it may include more than one gene

Promoter and terminators define the

unit

Upstream

sequences in the opposite direction from expression

Downstream

sequences proceeding farther in the direction of expression within the transcription unit

primary transcript

the original unmodified RNA product corresponding to a transcription unit

Transcription occurs by base pairing in a

“bubble” of unpaired DNA

RNA polymerase separates the two strands of DNA in a transient “bubble” and uses

one strand as a template to direct synthesis of a complementary sequence of RNA

The bubble is 12 to 14 bp, and the RNA-DNA hybrid within the bubble is

8 to 9 bp

RNA synthesis occurs in the

transcription bubble

RNA polymerase surrounds

the bubble

Transcription has ____ stages

three

RNA polymerase binds to

a promoter site on DNA to form a closed complex

RNA polymerase initiates

transcription (initiation) after opening the DNA duplex to form a transcription bubble (the open complex)

RNA polymerase catalyzes

transcription

During elongation

the transcription bubble moves along DNA and the RNA chain is extended in the 5’ to 3’ direction by adding nucleotides to the 3’ end.

Transcription stops (termination) and the DNA duplex reforms when

RNA polymerase dissociates at a terminator site

Bacterial RNA Polymerase consists of

multiple subunits

Holoenzyme

The RNA polymerase form that is competent to initiate transcription. It consists of the five subunits of the core enzyme and sigma factor.

RNA polymerase has _ types of subunit

4

Catalysis derives from the

B and B’ subunits

CTD (C-terminal domain)

The domain of RNA polymerase that is involved in stimulating transcription by contact with regulatory proteins

RNA Polymerase holoenzyme consists of

the core enzyme and sigma factor

Bacterial RNA polymerase can be divided into

the a2BB’w core enzyme that catalyzes transcription and the sigma subunit that is required only for initiation

Sigma factor changes the DNA-bidning properties of RNA polymerase so that

its affinity for general DNA is reduced and its affinity for promoter is increased

Sigma factor controls

specificity

The rate at which RNA polymerase binds to promoters can be too fast to be accounted for by

simple diffusion

RNA polymerase binds to

random sites on DNA and exchanges them with other sequences until a promoter is found.

Proposed mechanisms for how RNA polymerase finds a promoter

• sliding

• intrasegment transfer “hopping”

The holoenzyme goes through

transitions in the process of recognizing and escaping from promoters

Ternary complex

The complex in initiation of transcription that consists of RNA polymerase and DNA as well as a dinucleotide that represents the first two bases in the RNA product

There may be a cycle of ______ __________ before the enzyme moves to the next phase

abortive initiations

Sigma factor is usually released from RNS polymerase when the

nascent RNA chain reaches ~10 bases in length

Sigma factor controls

binding to DNA by recognizing specific sequences in promoters

conserved sequence

sequences in which many examples of a particular nucleic acid or protein are compared and the same individual bases or amino acids are always found at particular locations

A promoter is defined by the presence of short _________ _________ at specific locations

consensus sequences

The promoter consensus sequences usually consist of

a purine at the startpoint, a hexamer with a sequence close to TATAAT centered at ~ -10 (-10 element or TATA box), and another hexamer with a sequence similar to TTGACA at ~ -35 (-35 element)

Individual promoters usually differ from the consensus at

one or more positions

Promoter efficiency can be affected by

additional elements as well; it can be increased of decreased by mutation

• UP element

• Down mutations

UP element

A sequence in bacteria adjacent to the promoter, upstream of the -35 element, that enhances transcription

DNA elements and RNA polymerase modules contributing to promoter recognition by

sigma factor

Down mutations to decrease promoter efficiency usually

decrease conformance to the consensus sequences, whereas up mutations have the opposite effect

Up mutations

have the opposite effect of down mutations, increases conformance to the consensus sequences

Mutations in the -35 sequence can affect

initial binding of RNA polymerase

Mutations in the -10 sequence can affect

binding or the melting reaction that converts a closed to an open complex

Footprinting

a technique for identifying the site on DNA bound by some protein by virtue of the protection of bonds in this region against attack by nucleases

• a high resolution method for characterizing RNA polymerase-Promoter and DNA-Protien interactions in general

A protein protects a series of bonds against nuclease attack

The consensus sequences at -35 and -10 provide

most of the contact points for RNA polymerase in the promoter.

The point of contact lie primarily on

one face of the DNA

RNA polymerase contacts

one face of DNA

Interactions between sigma factors and Core RNA polymerase change during

promoter escape

A domain in sigma occupies

the RNA exit channel and must be displaced to accommodate RNA synthesis

Abortive initiations usually occur

before the enzyme forms a true elongation complex

Sigma factor is usually released from RNA polymerase by

the time the nascent RNA chain reaches ~10 nt in length

A model for enzyme movement is suggested by the

crystal structure

DNA moves through a channel in RNA polymerase and

makes a sharp turn at the active site

Changes in conformations of certain flexible modules within the enzyme control

the entry of nucleotides to the active site

DNA is forced to make a turn at the active site by

a wall of protein

Bacterial RNA polymerase terminates at

discrete sites

There are two classes of terminators

• intrinsic terminators

• rho-dependent terminators

intrinsic terminators

those recognized solely by RNA polymerase itself without the requirement for any cellular factors

rho-dependent terminators

requires a cellular protein called rho

The DNA sequences required for termination are located

upstream of the terminator sequence

Intrinsic termination requires

recognition of a terminator sequence in DNA that codes for a hairpin structure in the RNA product

The signals for termination lie mostly within sequences

already transcribed by RNA polymerase

Termination relies on

scrutiny of the template and/or the RNA product that the polymerase is transcribing

An intrinsic terminator has two features

Single-stranded U-run & G-C-rich region in stem

Readthrough

it occurs at transcription or translation when RNA polymerase or the ribosome, respectively, ignores a termination signal because of a mutation of the template or the behavior of an accessory factor

Antitermination

A mechanism of transcriptional control in which termination is prevented at a specific terminator site, allowing RNA polymerase to read into the genes beyond it.

Rho factor is a protein that

binds to nascent RNA and tracks along the RNA to interact with RNA polymerase and release it from the elongation complex

rut

an acronym for rho utilization site, the sequence of RNA that is recognized by the rho termination factor

Rho terminates transcription

1. RNA polymerase transcribes DNA

2. Rho attaches to rut site on RNA

3. Rho translocating along RNA

4. RNA polymerase pauses at hair pin and rho catches up

5. Rho unwinds DNA-RNA hybrid

6. Termination: all components released

Polarity

The effect of a mutation in one gene in influencing the expression (at transcription or translation) of subsequent genes in the same transcription unit.

Antitermination complex

Proteins that allow RNA polymerase to transcribe through certain terminator sites

Rho can terminate when

a nonsense mutation removes ribosomes

Wilt Type transcription

1. Ribosomes pack mRNA behind RNA polymerase

2. Ribosomes impede rho attachment and/or movement

3. Rho attaches but ribosomes impede its movement

4. Transcription continues

Nonsense mutant transcription

1. Ribosomes pack mRNA behind RNA polymerase

2. Ribosomes dissociate at mutation

3. Rho obtains access to RNA polymerase

4. Transcription terminates prematurely

Supercoiling is

an important feature of transcription

Negative supercoiling

increases the efficient of some promoters by assisting the melting reaction

Transcription generates

positive supercoils ahead of the enzyme and negative supercoils behind it. and these must be removed by gyrase and topoisomerase

Transcription changes

DNA structure

Negative supercoils → Transcribing DNA → Overwound (Positive supercoils)

Topoisomerase relaxes

negative supercoils

Gyrase introduces

positive supercoils

Competition for sigma factors can

regulate initiation

E.coli has _______ sigma factors

seven, each of which causes RNA polymerase to initiate at a set of promoters defined by specific -35 and -10 sequences

Sigma factor controls

promoter recognition

Holoenzyme with σ⁷⁰ recognizes one set of promoters

Substitution of sigma factor causes enzyme to recognize a different set of promoters

The activities of the different sigma factors are regulated by

different mechanisms

anti-sigma factor

a protein that binds to a sigma factor to inhibit its ability to utilize specific promoters

Heat Shock response

a set of loci that is activated in response to an increase in temperature that causes proteins to denature (and other abuses to the cell)

• all organisms have this response

• the gene products usually include chaperones that act on denatured proteins

sigma factors may be

organized into cascades

A cascade of sigma factors is created

when one sigma factor is required to transcribe the gene coding for the next sigma factor

The early genes of phage SPO1 are transcribed by

host RNA polymerase