Chapter 19

Prokaryotic Transcription

19.1 Introduction

•  RNA synthesis (transcription) occurs in 5′ - 3′ direction on a template strand (antisense strand) that is 3′ to 5′.

•  Coding strand (sense strand) – the DNA strand that has the same sequence as the mRNA.

•  RNA polymerase – an enzyme that synthesizes RNA using a DNA template (a DNA-dependent RNA polymerase).

•  Primary transcript: initial product of transcription, original unmodified RNA product

5’ 5’

5’ 5’ 3’

3’ 3’

3’

Gene A Gene B

Gene A Gene B 5’ 5’ 3’

3’ 5’

5’ 3’

3’

19.1 Introduction

•  RNA synthesis (transcription) occurs in 5′ - 3′ direction on a template strand (antisense strand) that is 3′ to 5′.

•  Coding strand (sense strand) – the DNA strand that has the same sequence as the mRNA.

•  RNA polymerase – an enzyme that synthesizes RNA using a DNA template (a DNA-dependent RNA polymerase).

•  Primary transcript: initial product of transcription, original unmodified RNA product

5’ 5’

5’ 5’ 3’

3’ 3’

3’

Gene A Gene B

Gene A Gene B 5’ 5’ 3’

3’ 5’

5’ 3’

3’

19.1 Introduction

•  promoter – A region of DNA where RNA polymerase binds to initiate transcription.

•  terminator – A sequence of DNA that causes RNA polymerase to terminate transcription.

•  transcription unit – A DNA sequence from promoter to terminator; it may include more than one gene (= poly-cistronic).

•  startpoint – The position on DNA corresponding to the first base incorporated into RNA. It is given the value of +1; immediate upstream of +1 is -1 (no 0).

19.1 Introduction

Figure 19.02

•  Downstream: after the start site (+1). •  Upstream: before the start site (begins with -1).

19.2 Transcription Occurs by Base

Pairing in a “Bubble” of Unpaired DNA

•  The length of the bubble is ~12 to 14 bp, and the length of RNA-DNA hybrid within it is ~8 to 9 bp.

•  Transcription rate: 40 – 50 nt/sec

•  Translation rate: 15 aa/sec

•  DNA replication rate: ~800 bp/sec

Figure 19.03

19.3 The Transcription Reaction Has Three Stages

•  initiation: template recognition and open complex (“bubble”)formation; typically short RNA (<10 nt) is released (abortive transcription) until elongation starts. RNA polymerase does not move.

•  elongation: the transcription bubble moves along DNA and the RNA chain is extended in the 5′ to 3′ direction.

•  termination: RNA polymerase dissociates and RNA is released.

Figure 19.06

19.4 Bacterial RNA Polymerase Consists of the Core Enzyme and Sigma Factor

•  Only one kind of RNA polymerase in E. coli (~13,000 molecules per E. coli).

•  Bacterial RNA polymerase holoenzyme can be divided into an core enzyme (α2ββ′ω) that catalyzes transcription and a sigma (σ) subunit that is required only for initiation.

•  Sigma factor changes the DNA-binding properties of RNA polymerase so that its affinity for general DNA is reduced and its affinity for promoters is increased = sigma factor is required for promoter binding of RNA polymerase.

•  Sigma factor does not bind promoters by itself.

19.4 Bacterial RNA Polymerase

Consists of the Core Enzyme and Sigma

Factor

Figure 19.07

•  Half-life of core enzyme – any DNA complex is ~1 hour and sigma factor reduces it to <1 second.

•  Sigma factor is responsible for stable interaction of holoenzyme and promoter (half-life of several hours).

19.5 How Does RNA Polymerase Find Promoter Sequences?

Figure 19.08

19.6 Sigma Factor Controls Binding to Promoters

•  RNA polymerase binds to the promoter as a closed complex in which the DNA remains double stranded.

•  RNA polymerase then separates the DNA strands to form an open complex.

•  RNA polymerase incorporates the first two nucleotides and form ternary complex (RNA polymerase + DNA + RNA), which can grow up to ~9 nucleotides-long RNA without movement of RNA polymerase.

•  There may be a cycle of abortive initiations before the enzyme moves out of promoter.

•  Promoter clearance: RNA polymerase leaves promoter and start elongation.

•  Sigma factor may be released from RNA polymerase core enzyme when the nascent RNA is elongated.

•  Alternatively, its association may be modified in a way to allow promoter clearance.

Figure 19.10

19.6 Sigma Factor Controls Binding to Promoters

19.6 Sigma Factor Controls Binding to Promoters

•  A change in association between sigma factor and core enzyme changes binding affinity for DNA, so that core enzyme can move along DNA.

•  Sigma factor is not required for elongation.

Figure 19.11

19.7 Promoter Recognition Depends on Consensus Sequences

•  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.

•  consensus sequences – sequences that represent nucleotides or amino acids most often present at a particular position.

19.7 Promoter Recognition Depends on Consensus Sequences

•  The promoter consensus sequences consist of a purine at the startpoint, -10 element (TATAAT; Pribnow box), and another hexamer centered at –35 (-35 element; -35 box).

•  Individual promoters usually differ from the consensus at one or more positions.

•  Promoter efficiency can be affected by additional elements as well; space between -10 and -35 elements, -35 element upstream (e.g., UP element).

19.7 Promoter Recognition Depends on Consensus Sequences

Figure 19.12

Sigma factor RNA polymerase α subunit

19.8 Promoter Efficiencies Can Be Increased or Decreased by Mutation

•  Down mutations that decrease promoter efficiency usually decrease conformance to the consensus sequences, whereas up mutations have the opposite effect.

•  Mutations in the –35 sequence can affect initial binding of RNA polymerase (i.e., closed complex formation). However, they do not affect the rate of open complex formation.

•  Mutations in the –10 sequence affect formation of closed complex or open complex, or both.

•  -10 and -35 element interact with sigma factor. •  -10 element is crucial for “melting”

19.9 Multiple Regions in RNA Polymerase Directly Contact Promoter DNA

•  σ70 changes its structure to expose its DNA-binding regions when it associates with core enzyme.

•  N-terminal region masks DNA-binding domains (DBDs); however, DBDs are exposed upon binding to core enzyme.

Figure 19.15

19.11 Bacterial Transcription Termination

•  Terminator (t): DNA sequence that ends transcription.

•  Actual signal for transcription termination often lies in RNA.

•  Most common signal is a hairpin structure in the RNA product. Figure 19.19

19.12 Intrinsic Termination Requires a

Hairpin and U-Rich Region

•  Intrinsic terminators do not require auxiliary protein factors.

•  They consist of a G-C-rich hairpin in the RNA product followed by a U-rich region in which termination occurs.

Figure 19.20

•  U-rich region destabilizes RNA-DNA hybrid when RNA polymerase pauses at the hairpin.

19.13 Rho Factor Is a Site-Specific Terminator Protein

•  Rho factor is a terminator protein and hexameric helicase.

•  Rho binds to a rut site on a nascent RNA and tracks along the RNA to release it from the RNA-DNA hybrid structure.

•  rut – An acronym for rho utilization site, the sequence of RNA that is recognized by the rho termination factor. It is upstream of the site of termination. Common feature is C-rich sequence.

Figure 19.21

19.15 Substitution of Sigma Factors May Control Initiation

•  E. coli has seven sigma factors, each of which causes RNA polymerase to initiate at a set of promoters defined by specific –35 and –10 sequences. E. coli responds to environmental changes by activating specific sigma factors.

Figure 19.26

19.16 Antitermination May Be a Regulated Event •  Antitermination: inhibition of transcription termination.

Figure 19.28

Antitermination protein

•  Antitermination results in readthrough, which can generate transcripts containing more than one gene.

19.17 The Cycle of Bacterial Messenger RNA

•  coupled transcription/translation in bacteria, as ribosomes begin translating an mRNA before its synthesis has been completed.

•  Multiple ribosomes move along mRNA polysomes.

•  Bacterial mRNA is unstable (degraded from 5’ end) and has a half-life of only a few minutes.

•  3’ end is generated when transcription terminates.

Figure 19.30

19.17 The Cycle of Bacterial Messenger RNA •  Multiple mRNAs are undergoing synthesis simultaneously. •  Each mRNA carries many ribosomes shown as large dots in

the figure.

Figure 19.31

19.17 The Cycle of Bacterial Messenger RNA

•  nascent RNA – An RNA chain that is still being synthesized, so that its 3' end is paired with DNA where RNA polymerase is elongating.

•  monocistronic mRNA – mRNA that encodes one protein.

•  A bacterial mRNA may be polycistronic in having several coding regions that represent different cistrons.

19.17 The Cycle of Bacterial Messenger RNA

•  5′ UTR (untranslated region) – The untranslated sequence upstream from the coding region of an mRNA.

•  3′ UTR – The untranslated sequence downstream from the coding region of an mRNA.

•  5’ UTR and 3’ UTR regulate translation of mRNA.

Figure 19.32