Biochemistry 111. Carl Parker x A Braun

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1 Biochemistry 111 Carl Parker x A Braun

2 Central Dogma of Molecular Biology

3 DNA-Dependent RNA Polymerase Requires a DNA Template Synthesizes RNA in a 5 to 3 direction Requires ribonucleoside tri-phosphates rntps Requires a divalent cation Mg The enzyme is very large with 4 subunits (bacterial enzyme)

4 Structure of E. coli RNAP

5 6.1 RNA Polymerase Structure By 1969 SDS-PAGE of RNA polymerase from E. coli had shown several subunits 2 very large subunits are β (150 kd) and β (160 kd) Sigma (σ) at 70 kd Alpha (α) at 40 kd 2 copies present in holoenzyme Omega (w) at 10 kd Was not clearly visible in SDS-PAGE, but seen in other experiments Not required for cell viability or in vivo enzyme activity Appears to play a role in enzyme assembly 6-5

6 Activity of core vs holoenzyme.

7 Eσ and Ecore have different properties Both will transcribe a heterogenous sheared DNA template Only the holoenzyme can transcribe an intact specific template Ecore has the catalytic activity The sigma subunit plays a role in template selection

8 Binding of RNA Polymerase to Promoters How tightly does core enzyme v. holoenzyme bind DNA? Experiment measures binding of DNA to enzyme using nitrocellulose filters Holoenzyme binds filters tightly Core enzyme binding is more transient 6-8

9 Sigma forces rapid dissociation and facilitates stable complex

10 Results of Hinkle and Chamberlain s experiment E/DNA Ratio % stable # stable complexes % loose # loose complexes total 8/ / /

11 Closed and Open Promoter Complexes

12 Shapes of E.coli RNAP core (a) holoenzyme (b)

13 Model for RNAP interaction with promoter DNA

14 Summary The σ-factor allows initiation of transcription by causing the RNA polymerase holoenzyme to bind tightly to a promoter This tight binding depends on local melting of the DNA to form an open promoter complex and is stimulated by σ The σ-factor can therefore select which genes will be transcribed 6-14

15 Core Promoter Elements There is a region common to bacterial promoters described as 6-7 bp centered about 10 bp upstream of the start of transcription = -10 box Another short sequence centered 35 bp upstream is known as the -35 box Comparison of thousands of promoters has produced a consensus sequence (or most common sequence) for each of these boxes 6-15

16 Promoter Strength Consensus sequences: -10 box sequence approximates TATAAT -35 box sequence approximates TTGACA Mutations that weaken promoter binding: Down mutations Increase deviation from the consensus sequence Mutations that strengthen promoter binding: Up mutations Decrease deviation from the consensus sequence 6-16

17 UP Element The UP element is upstream of the core promoter, stimulating transcription by a factor of 30 UP is associated with 3 Fis sites which are binding sites for the transcription-activator protein Fis, not for the polymerase itself 6-17

18 The rrn P1 Promoter

19 6.3 Transcription Initiation Transcription initiation was assumed to end as RNA polymerase formed 1 st phosphodiester bond Carpousis and Gralla found that very small oligonucleotides (2-6 nt long) are made without RNA polymerase leaving the DNA Abortive transcripts such as these have been found up to 10 nt 6-19

20 Synthesis of short transcripts by RNAP bound to a promoter

21 Stages of Transcription Initiation

Sigma Stimulates Initiation of Transcription In this first experiment stimulation by σ appears to cause both initiation and elongation Or stimulating

22 Sigma Stimulates Initiation of Transcription In this first experiment stimulation by σ appears to cause both initiation and elongation Or stimulating initiation by σ provides more initiated chains for core polymerase to elongate Further experiments by the same group proved that σ does not stimulate elongation 6-22

23 Reuse of σ During initiation σ can be recycled for additional use with a new core polymerase The core enzyme can release σ which is then free to associate with another core enzyme 6-23

24 Fluorescence Resonance Energy Transfer The σ-factor changes its relationship to the core polymerase during elongation It may not dissociate from the core but actually shift position and become more loosely bound to core To answer this question Fluorescence Resonance Energy Transfer (FRET) was used as it relies on two fluorescent molecules that are close enough together to engage in transfer of resonance energy FRET allows the position of σ relative to a site on the DNA to be measured without using separation techniques that might displace σ from the core enzyme 6-24

25 FRET Assay for σ Movement Relative to DNA 6-25

26 Models for the σ-cycle The obligate release version of the σ-cycle model arose from experiments performed by Travers and Burgess that proposed the dissociation of σ from core as polymerase undergoes promoter clearance and switches from initiation to elongation mode The stochastic release model proposes that σ is indeed released from the core polymerase but that there is no discrete point of release during transcription and that the release occurs at random - a preponderance of evidence favors this model 6-26

27 Structure and Function of σ Genes encoding a variety of σ-factors have been cloned and sequenced There are striking similarities in amino acid sequence clustered in 4 regions Conservation of sequence in these regions suggests important function All of the 4 sequences are involved in binding to core and DNA 6-27

28 Homologous Regions in Bacterial σ Factors 6-28

29 E. coli σ 70 Four regions of high sequence similarity are indicated Specific areas that recognize the core promoter elements are the -10 box and 35 box 6-29

30 Region 1 Role of region 1 appears to be in preventing σ from binding to DNA by itself This is important as σ binding to promoters could inhibit holoenzyme binding and thereby inhibit transcription Region 2 This region is the most highly conserved of the four There are four subregions 2.1 to recognizes the promoter s -10 box The 2.4 region appears to be α-helix 6-30

31 Regions 3 and 4 Region 3 is involved in both core and DNA binding Region 4 is divided into 2 subregions This region seems to have a key role in promoter recognition Subregion 4.2 contains a helix-turn-helix DNAbinding domain and appears to govern binding to the -35 box of the promoter 6-31

32 Specific interactions between the sigma subunit and promoter elements

33 Role of α-subunit in UP Element Recognition RNA polymerase itself can recognize an upstream promoter element, UP element While σ-factor recognizes the core promoter elements, what recognizes the UP element? It appears to be the α-subunit of the core polymerase 6-33

Modeling the Function of the C-Terminal Domain RNA polymerase binds to a core promoter via its σ-factor, no help from C-terminal domain of α-subunit Binds to a promoter with an

34 Modeling the Function of the C-Terminal Domain RNA polymerase binds to a core promoter via its σ-factor, no help from C-terminal domain of α-subunit Binds to a promoter with an UP element using σ plus the α- subunit C-terminal domains (CTD) Results in very strong interaction between polymerase and promoter This produces a high level of transcription 6-34

35 The Elongation Complex

36 6.5 Termination of Transcription When the polymerase reaches a terminator at the end of a gene it falls off the template and releases the RNA There are 2 main types of terminators Intrinsic terminators function with the RNA polymerase by itself without help from other proteins Other type depends on auxiliary factor called rho (ρ), these are rho or ρ-dependent terminators 6-36

37 Inverted Repeats and Hairpins The repeat at right is symmetrical around its center shown with a dot A transcript of this sequence is selfcomplementary Bases can pair up to form a hairpin as seen in the lower panel 6-37

Model of Intrinsic Termination Bacterial terminators act by: Base-pairing of the transcript to destabilize RNA-DNA hybrid Causes hairpin to form This

38 Model of Intrinsic Termination Bacterial terminators act by: Base-pairing of the transcript to destabilize RNA-DNA hybrid Causes hairpin to form This causes transcription to pause a string of U s incorporated just downstream of hairpin to destabilize the hybrid and the RNA falls off the DNA template 6-38

39 Rho-Dependent Termination Rho caused depression of the ability of RNA polymerase to transcribe phage DNAs in vitro This depression was due to termination of transcription After termination, polymerase must reinitiate to begin transcribing again 6-39

40 Rho Affects Chain Elongation There is little effect of rho or ρ on transcription initiation, if anything it is increased The effect of rho or ρ on total RNA synthesis is a significant decrease This is consistent with action of rho or ρ to terminate transcription forcing time-consuming reinitiation 6-40

41 Mechanism of Rho No string of T s in the ρ- dependent terminator, just inverted repeat to hairpin Binding to the growing transcript, ρ follows the RNA polymerase It catches the polymerase as it pauses at the hairpin Releases transcript from the DNA-polymerase complex by unwinding the RNA-DNA hybrid 6-41

42 Summary Using the trp attenuator as a model rho-independet terminator revealed two important features: 1 - an inverted repeat that allows a hairpin to for at the end of the transcript 2 - a string of T s in the nontemplate strand that results in a string of weak ru-da base pairs holding the transcript to the template strand Rho-dependent terminators consist of an inverted repeat, which can cause a hairpin to form in the transcript but no string of T s 6-42

43 Rho-independent termination

44 Rho-dependent termination

RNA Expression of the information in a gene generally involves production of an RNA molecule transcribed from a DNA template. RNA differs from DNA

RNA Expression of the information in a gene generally involves production of an RNA molecule transcribed from a DNA template. RNA differs from DNA that it has a hydroxyl group at the 2 position of the