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Biochemistry 201 Biological Regulatory Mechanisms Transcription and Its Regulation January 22 –Mechanism of Transcription Initiation January 24– Mechanism of Transcription Elongation January 28– Control of Transcription in Bacteria January 31– Control of Transcription in Eukaryotes

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Biochemistry 201

Biological Regulatory Mechanisms

Transcription and Its Regulation

January 22 –Mechanism of Transcription Initiation

January 24– Mechanism of Transcription Elongation

January 28– Control of Transcription in Bacteria

January 31– Control of Transcription in Eukaryotes

Mechanism of Transcription Initiation

References

I. General

Chapter 12 of Molecular Biology of the Gene 6th Edition (2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine, M, Losick, R. 377-414.

2.

2. Reviews

Murakami KS, Darst SA. (2003) Bacterial RNA polymerases: the wholo story. Curr Opin Struct Biol 13:31-9.

Campbell, E, Westblade, L, Darst, S., (2008) Regulation of bacterial RNA polymerase factor activity: a structural perspective. Current Opinion in Micro. 11:121-127

Herbert, KM, Greenleaf, WJ, Block, S. (2008) Single-Molecule studies of RNA polymerase: Motoring Along. Annu Rev Biochem. 77:149-76.

Werner, Finn and Dina Grohmann (201). Evolution of multisubunit RNA polymerases in the three domains of life. Nature Rev. Microbiology 9: 85-98

3. Studies of Transcription Initiation

Roy S, Lim HM, Liu M, Adhya S. (2004) Asynchronous basepair openings in transcription initiation: CRP enhances the rate-limiting step. EMBO J. 23:869-75.

Sorenson MK, Darst SA. (2006).Disulfide cross-linking indicates that FlgM-bound and free sigma28 adopt similar conformations. Proc Natl Acad Sci U S A. 103:16722-7.


Young BA, Gruber TM, Gross CA. (2004) Minimal machinery of RNA polymerase holoenzyme sufficient for promoter melting. Science.303:1382-1384

*Kapanidis, AN, Margeat, E, Ho, SO,.Ebright, RH. (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science.314:1144-1147.

Revyakin A, Liu C, Ebright RH, Strick TR (2006) Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science. 314: 1139-43.

Murakami KS, Masuda S, Campbell EA, Muzzin O, Darst SA (2002). Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex. Science. 296:1285-90.

Kostrewa D, Zeller ME, Armache KJ, Seizl M, Leike K, Thomm M, Cramer P.(2009) RNA polymerase II-TFIIB structure and mechanism of transcription initiation. Nature. 462:323-30.

Discussion Paper

**Feklistov A and Darst, SA (2011) Structural basis for Promoter -10 Element recognition by the Bacterial RNA Polymerase s Subunit. Cell147: 1257 – 1269

Accompanying preview: Liu X, Bushnell DA and Kornberg RD ( 2011) Lock and Key to Transcription:

s –DNA Interaction. Cell: 147: 1218-1219

***Paul BJ, Barker MM, Ross W, Schneider DA, Webb C, Foster JW, Gourse RL. (2004) DksA: a critical component of the transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP.

Cell. 6:311-22.

The accompanying minireview is helpful

Nickels, B.E. and Hochschild, A. (2004) Regulation of RNA Polymerase through the Secondary Channel. Cell118:281-284


  • Key Points RNA polymerase holoenzyme sufficient for promoter melting.

  • 1. Multisubunit RNA polymerases are conserved among all organisms

    • 2. RNA polymerases cannot initiate transcription on their own. In bacteria s70is required to initiate transcription at most promoters. Among other functions, it recognizes the key features of most bacterial promoters, the -10 and -35 sequences.

    • 2. E. coli RNA polymerase holoenzyme, (core + s) finds promoter sequences by sliding along DNA and by transfer from one DNA segment to another. This behavior greatly speeds up the search for specific DNA sequences in the cell and probably applies to all sequence-specific DNA-binding proteins.

    • 3. Transcription initiation proceeds through a series of structural changes in RNA polymerase,s70and DNA.

    • 4. A key intermediate in E. coli transcription initiation is the open complex, in which the RNA polymerase holoenzyme is bound at the promoter and ~12 bp of DNA are unwound at the transcription startpoint. Open complex formation does not require nucleoside triphosphates. Its presence can be monitored by a variety of biochemical and structural techniques.

    • 5. Recognition of the -10 element of the promoter DNA is coupled with strand separation

    • 6. When the open complex is given NTPs, it begins the ‘abortive initiation’ phase, in which RNA chains of 5-10 nucleotides are continually synthesized and released.

  • 7. Through a “DNA scrunching” mechanism the energy captured during synthesis of one of these short

  • transcripts eventually breaks the enzyme loose from its tight connection to the promoter DNA, and it begins

  • the elongation phase.

  • 7. Aspects of the mechanism of initiation are likely to be conserved in eukaryotic RNA polymerase


  • Transcription is important

    transcription RNA polymerase holoenzyme sufficient for promoter melting.

    (RNA processing)

    snRNAs

    miRNAs

    rRNAs

    mRNAs

    Other non-coding RNAs

    (e.g. telomerase RNA)

    translation

    proteins

    Transcription is Important


    Transcription/Splicing/Translation Provide RNA polymerase holoenzyme sufficient for promoter melting.

    A Large Range of Protein Concentrations


    I. RNA polymerases RNA polymerase holoenzyme sufficient for promoter melting.


    Cellular RNA polymerases in RNA polymerase holoenzyme sufficient for promoter melting. all living organisms are evolutionary related

    Subunits of RNAP

    LUCA-Last universal common ancestor

    a common structural and functional frame work of transcription in the three domains of life


    Structure of RNAP in t RNA polymerase holoenzyme sufficient for promoter melting. he three domains

    Universally conserved

    Archaeal/eukaryotic

    Bacteria

    Archaea

    Eukarya

    Transcription

    Extra RNAP subunits provide interaction sites for transcription factors, DNA and RNA, and modulate diverse RNAP activities

    Werner and Grohmann (2011),

    Nature Rev Micro 9:85-98


    Evolutionary RNA polymerase holoenzyme sufficient for promoter melting. relationships of general transcription factors

    s

    Initiation

    s

    Gre

    Transcript cleavage

    Elongation

    LUCA may have had elongating, not initiating RNA polymerase


    II. Challenges in initiating transcription RNA polymerase holoenzyme sufficient for promoter melting.

    • RNAP is specialized to ELONGATE, not INITIATE

    2. Initiating RNAP must open DNA to permit transcription

    3. RNAP must leave promoter—abortive initiation


    The Initiating Form of RNA Polymerase RNA polymerase holoenzyme sufficient for promoter melting.


     RNA polymerase holoenzyme sufficient for promoter melting. 

    (1) The discovery of initiation factors

     factor is required for bacterial RNA polymerase to initiate transcription on promoters

    +

    

    '

    '

    KD ~ 10-9 M

    }

    }

    ‘holoenzyme’

    ‘core’

    Can elongate but cannot begin transcription at promoters

    Can begin transcription on promoters and can elongate


    B. RNA polymerase holoenzyme sufficient for promoter melting. Initial purification

    Lysate

    various fractionation steps

    (DEAE column, glycerol gradient etc)

    Active fractions identified by assay

    How was discovered (Burgess, 1969)

    A. Assay for RNA polymerase:

    *ATP

    CTP

    GTP

    UTP

    E.coli lysate

    Calf thymus DNA

    buffer

    Look for incorporation of *ATP into RNA chains


    lysate RNA polymerase holoenzyme sufficient for promoter melting.

    Improved fractionation

    phosphocellulose column

    2

    Activity (*ATP)

    CT DNA

    1

    Peak 1 Peak 2

    Fraction #

    '

     increases rate of initiation

    SDS gel analysis

    Assay:

    incorporationPATP

    Transcription

     DNA

    g 

    C. Improved purification of RNA polymerase:

    Labmate Jeff Roberts reported that the new, improved preparation of RNAP (peak 2) had no activity on  DNA

    salt

    OD 280

    Peak 1 restored activity


    There are several flavors of promoters RNA polymerase holoenzyme sufficient for promoter melting.

     and  recruit RNAP to promoter DNA

    (2) Bacterial promoters


    (3) RNA polymerase holoenzyme sufficient for promoter melting. s undergoes a large conformational change upon binding

    to RNA polymerase

    Free  doesn’t bind DNA  in holoenzyme positioned for DNA recognition

    Sorenson; 2006


    s RNA polymerase holoenzyme sufficient for promoter melting. is positioned for DNA recognition


    s RNA polymerase holoenzyme sufficient for promoter melting. is positioned to affect key activities of RNA polymerase


    Surprising structural similarity between the initiating forms of bacterial and eukaryotic RNAP


    The first two steps of Eukaryotic transcription forms of bacterial and eukaryotic RNAP

    TFB

    TBP

    Promoter

    In archae, TBP and TFB are sufficient for formation of the pre-initiation complex (PIC), suggesting that they are key to the mechanism of transcription initiation in eukaryotes

    Many archae have a proliferation of TBPs and TFBs, suggesting that

    they provide choice in promoters, akin to alternative s.


    TFIIB structure forms of bacterial and eukaryotic RNAP

    TFIIB has a central role in initiation similar to that of 

    Recruits Pol II to promoter: N-terminus binds

    Pol II; C terminus binds TBP and DNA

    Role in promoter opening; B linker mutants recruit PolII but cant strand open or initiate

    Role in selection of TSS ( Inr): B reader mutants

    Blocks elongating RNAchain: B reader

    Crystal structure of TFB + RNA polymerase--archae

    D Kostrewa et al.Nature462, 323-330 (2009) doi:10.1038/nature08548


    Topological similarities in forms of bacterial and eukaryotic RNAP/TFIIB binding to RNAP

    B ribbon (4):both bind flap tip helix

    B linker (2): both bind coiled -coil and rudder; both

    involved in strand opening

    B core (3)

    B reader ( 3.2): both in exit channel and near

    active site; start site selection

    D Kostrewa et al.Nature462, 323-330 (2009) doi:10.1038/nature08548

    TFIIB and  bound to RNA polymerase show surprising similarity. Analogously placed regions have similar functions


    Initiating RNAP must open DNA to permit transcription: forms of bacterial and eukaryotic RNAP

    Formation of the open complex


    NTPs forms of bacterial and eukaryotic RNAP

    KB

    Kf

    Elongating

    Complex

    Abortive

    Initiation

    R+P

    RPc

    RPo

    initial

    binding

    “isomerization”

    Steps in transcription initiation


    A detailed look at a prokaryotic promoter forms of bacterial and eukaryotic RNAP

    15-19nucleotides

    T

    T

    G

    A

    C

    A

    T

    A

    T

    A

    A

    T

    -35

    -10

    Sequence Logos

    -35 logo

    -10 logo


    Recognition of the prokaryotic promoter forms of bacterial and eukaryotic RNAP

    -35 logo

    -10 logo

    Helix-turn-helix in Domain 4

    Recognizes -35 as duplex DNA

    Is the -10 promoter element recognized as Duplex or SS DNA?


    Approach forms of bacterial and eukaryotic RNAP

    1. Determine a high resolution structure of s2 bound to non-template strand of the -10 element

    Schematic

    2. Determine whether this structure represents the “initial binding state” or endpoint state


    Promoter escape forms of bacterial and eukaryotic RNAP


    s forms of bacterial and eukaryotic RNAPis positioned to affect key activities of RNA polymerase


    Promoter escape and Abortive Initiation forms of bacterial and eukaryotic RNAP

    during abortive initiation, RNAP synthesizes many short transcripts, but reinitiates rapidly. How can the active site of RNAP move forward along the DNA while maintaining promoter contact?


    Förster (fluorescence) resonance energy transfer (FRET) allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution

    Experimental set-up for single molecule FRET: Single transcription complexes labeled with a fluorescent donor (D, green) and a fluorescent acceptor (A, red) are illuminated as they diffuse through a femtoliter-scale observation volume (green oval; transit time ~1 ms); observed in confocal microscope

    Using single molecule FRET to monitor movement of RNAP and DNA


    Three models for Abortive initiation allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution

    #1

    Predicts movement of both the RNAP leading and trailing edge relative to DNA

    #2

    Predicts expansion and contraction of RNAP

    #3

    Predicts expansion and contraction of DNA


    Initial transcription involves DNA scrunching allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution

    Open complex

    Lower E* peak is free DNA; higher E* peak is DNA in open complex; distance is shorter because RNAP induces DNA bending

    A. N. Kapanidis et al., Science 314, 1144 -1147 (2006)


    Initial transcription involves DNA scrunching allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution

    Open complex

    Abortive initiation complex

    Higher E* in Abortive initiation complex than open complex results from DNA scrunching


    Initial transcription involves DNA scrunching allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution

    Open complex

    Abortive initiation complex


    The energy accumulated in the DNA scrunched allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution “stressed intermediate could disrupt interactions between RNAP,  and the promoter, thereby driving the transition from initiation to elongation

    At a typical promoter, promoter escape occurs only after synthesis of an RNA product ~9 to 11 nt in length (1–11) and thus can be inferred to require scrunching of ~7 to 9 bp (N – 2, where N = ~9 to 11; Fig. 3C). Assuming an energetic cost of base-pair breakage of ~2 kcal/mol per bp (30), it can be inferred that, at a typical promoter, a total of ~14 to 18 kcal/mol of base-pair–breakage energy is accumulated in the stressed intermediate. This free energy is high relative to the free energies for RNAP-promoter interaction [~7 to 9 kcal/mol for sequence-specific component of RNAP-promoter interaction (1)] and RNAP-initiation-factor interaction [~13 kcal/mol for transcription initiation factor {sigma}70 (31)].


    Validation of the prediction that allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution  occlusion of the RNA exit channel promotes “abortive initiation”

    #1: transcription by holoenzyme with full-length 

    #2: transcription by holoenzyme with truncated at Region 3.2: lacks  in

    the RNA exit channel

    Murakami, Darst 2002


    s allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution is positioned to affect key activities of RNA polymerase


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