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Transcription and Its Regulation January 21 –Mechanism of Transcription Initiation January 23– Regulation of of Transcription Initiation January 27–Mechanism and regulation of Transcription Elongation January 30– In class discussion of problem set 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. 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- 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- Grunberg, S. and Steven Hahn (2013) Structural Insights into transcription initiation by RNA polymerase II. TIBS 38: 603-
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- *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. Cell 147: 1257 – 1269 Accompanying preview: Liu X, Bushnell DA and Kornberg RD ( 2011) Lock and Key to Transcription: s –DNA Interaction. Cell : 147: 1218-
Key Points
- Multisubunit RNA polymerases are conserved among all organisms
- RNA polymerases cannot initiate transcription on their own. In bacteria s^70 is 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.
- Transcription initiation proceeds through a series of structural changes in RNA polymerase, s 70 and DNA.
- 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.
- Recognition of the -10 element of the promoter DNA is coupled with strand separation
- 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.
- 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.
- Aspects of the mechanism of initiation are likely to be conserved in eukaryotic RNA polymerase
rRNAs snRNAs^ miRNAs
Other non-coding RNAs
(e.g. telomerase RNA)
mRNAs
translation
proteins
transcription
(RNA processing)
Transcription is Important
I. RNA polymerases
Cellular RNA polymerases in all living organisms are evolutionary related
A common structural and functional frame work of transcription in the
three domains of life
LUCA-Last universal common
ancestor
Subunits
of
RNAP
Eukaryotic Cells have three RNA polymerases
TYPE OF POLYMERASE GENES TRANSCRIBED RNA polymerase I 5.85, 18S, and 28S rRNA genes RNA polymerase II all protein-coding genes, plus snoRNA genes, miRNA genes, siRNA genes, and some snRNA genes RNA polymerase III tRNA genes, 5S rRNA genes, some snRNA genes and genes for other small RNAs The rRNAs are named according to their “S” values, which refer to their rate of sedimentation in an ultra-centrifuge. The larger the S value, the larger the rRNA.
Evolutionary relationships of general transcription factorsEvolutionary relationships of general transcription factors
s
Initiation s
Gre
Transcript cleavage
Elongation
LUCA may have had elongating, not initiating RNA polymerase
The Initiating Form of RNA Polymerase
‘holoenzyme ’ '
KD ~ 10
M
Can begin transcription on promoters and can elongate
Can elongate but cannot begin transcription at promoters factor is required for bacterial RNA polymerase to initiate transcription on promoters ' (1) The discovery of initiation factors
Labmate Jeff Roberts reported that the new, improved preparation of RNAP (peak 2) had no activity on DNA Peak 1 restored activity C. Improved purification of RNA polymerase: Improved fractionation lysate phosphocellulose column sal t
OD 280
1 2
Activity (*ATP)
CT DNA
Fraction #
SDS gel analysis
Peak 1 Peak 2
increases rate of initiation
g
Transcription
DNA
Assay:
incorporation
P ATP
- 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 is positioned for DNA recognition
Initiating RNAP must open DNA to permit transcription: Formation of the open complex
