Mechanisms of Transcription. School of Life Science Shandong University

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1 Mechanisms of Transcription School of Life Science Shandong University

2 Ch 12: Mechanisms of Transcription 1. RNA polymerase and the transcription cycle 2. The transcription cycle in bacteria 3. Transcription in eukaryotes 4. Transcription by RNA polymerase I and III in eukaryotes

3 Transcription of DNA into RNA

4 1. RNA polymerase and the transcription cycle 1.1 RNA polymerases come in different forms but share many features RNA polymerases performs essentially the same reaction in all cells; Bacteria have only a single RNA polymerase while in eukaryotic cells there are three: RNA Pol I, II and III RNA Pol II is the focus of eukaryotic transcription, because it is responsible for transcribing most genes, essentially all protein-encoding genes

5 RNA Pol I transcribes the large ribosomal RNA precursor gene RNA Pol III transcribes trna gene, some small nuclear RNA genes and the 5S rrna genes

7 Comparison of the crystal structures of prokaryotic and eukaryotic RNA polymerases ω β T. aquatics S. cerevisiae

8 1.2 Transcription by RNA polymerase proceeds in steps: initiation, elongation, and termination

9 1.3 Transcription initiation involves three defined steps Formation of a closed complex through binding of RNA polymerase to a promoter The promoter-rna polymerase complex undergoes structural changes and transits to an open complex (transcription bubble with ~14 bp [+3 to -11] separation of DNA) Initial transcription followed by promoter escape: a transcript longer than 10 nt would enable the formation of a stable ternary complex.

10 Elongation: RNA polymerase performs a range of tasks in addition to RNA synthesis: unwinds the DNA in front, re-anneals it behind, and proofreading. Only 8-9 nt remain base-paired with the DNA template Termination: Specific and well-defined DNA sequences may trigger termination

11 2. The transcription cycle in bacteria RNA polymerase holoenzyme in E. coli RNA polymerase core enzyme (α 2 ββ ω) + σ factor = holoenzyme which initiates transcription at promoters in vivo RNA polymerase holoenzyme from Thermus aquaticus

12 2.1 Bacterial promoters have certain defining features Two conserved sequences, each of 6 bp, separated by a nonspecific stretch of bp; The two defined sequences are centered, respectively, at -10 and -35 bp upstream the start site (+1);

13 Features of bacterial σ 70 promoters UP-element, Increase polymerase binding No -35, extended -10 discriminator

14 2.2 The σ factor mediates binding of polymerase to promoter σ factor The -10 and -35 regions are recognized by regions 2 and 4 of σ factor;

15 σ and α subunits recruit RNA polymerase core enzyme to the promoter αctd: C-terminal of α subunit recognize UP-element

16 2.3 Transition to the open complex involves structural changes in RNA polymerase and in the promoter DNA RNA polymerase can initiate a new RNA chain without a primer.

17 2.4 During initial transcription, RNA polymerase remains stationary and pulls downstream DNA into itself Three models for explaining the initial transcription: Kapanidis AN et al. Science 2006

18 2.5 Promoter escape involves breaking polymerasepromoter and polymerase core σ interactions: The transcript with the length of 10 or more nucleotides cannot be accommodated within the region where it hybridizes to the DNA and must start threading into the RNA exit channel. Region 3/4 linker of σ factor mimics RNA lying in the middle of the RNA exit channel, and must be ejected from that location. The ejection probably accounts for the more weak association of σ with the elongating enzyme than it is with the open complex;

Scrunching is reversed upon escape, the DNA unwound during scrunching is rewound, with concomitant collapse of the transcription bubble from a size of 22-24 nt back

19 Scrunching is reversed upon escape, the DNA unwound during scrunching is rewound, with concomitant collapse of the transcription bubble from a size of nt back down to nt; Scrunching is a way to store and mobilize energy enabling polymerase to break free of the promoter and dislodge σ factor Scrunching is reversed upon escape

20 2.6 The elongating polymerase is a processive machine that synthesizes and proofreads RNA DNA enters the polymerase between the pincers; Strand separation in the catalytic cleft; NTP addition; RNA product spooling out (Only 8-9 nt of the growing RNA remain base-paired with the DNA template at any given time) The enzyme adds one nucleotide at a time to the growing RNA transcript. DNA strand annealing in behind

21 The elongating polymerase proofreads RNA Pyrohosphorolytic ( 焦磷酸键解 )editing: RNA polymerase catalyzes the removal of an incorrectly inserted ribonucleotide by reincorporation of ppi; Hydrolytic ( 水解 )editing: the enzyme backtracks ( 后退 ) by one or more nucleotides and removes the errorcontaining sequence. This is stimulated by Gre factor, a elongation stimulation factor.

22 As 1 bp is separated ahead of the processing enzyme, 1 bp is formed behind it. Hydrolytic editing

23 2. 7 Transcription is terminated by signals within the RNA sequences: Terminators trigger the dissociation of the elongating polymerase from the DNA and release the RNA chain it has made. Terminators come in two types in bacteria Rho-dependent and Rho independent;

24 Rho-dependent terminators have ill-defined RNA elements (rut sites: ~40 nt with no secondary structure and rich in C) for Rho to work Rho factor: RNA (rut sites) binding ; ATPase, use the energy derived from ATP hydrolysis to induce termination. fails to bind any transcript being translated

25 Rho-independent termination (intrinsic terminators ): Consist of two sequence elements an inverted repeat (20 nt) followed by a stretch of about A:T base pairs; Function in the RNA rather in the DNA

26 3. Transcription in eukaryotes Comparison with transcription in Prokaryotes: Prokaryotes: one polymerase transcribes all genes Eukaryotes: three polymerases transcribe different class of genes Prokaryotes: σ factors Eukaryotes: general transcription factors (GTFs, 通用转录因子 ) are all that are required for promoter-specific transcription initiation. Additional factors including regulatory proteins, Mediator complex, and chromatin-modifying enzymes are required for in vivo initiation.

3.1 RNA polymerase II core promoters are made up of combinations of four different sequence elements Core promoter (40-60 bp) is the minimal set of sequence elements required for accurate

27 3.1 RNA polymerase II core promoters are made up of combinations of four different sequence elements Core promoter (40-60 bp) is the minimal set of sequence elements required for accurate transcription initiation by Pol II machinery as measured in vitro. Extending either upstream or downstream from the transcription start site (+1) Typically, a promoter includes some subset of these elements. Pol II core promoter

28 Regulatory sequences are typically upstream of core promoter, and required for efficient transcription in vivo. Can be located tens or even hundreds of kilobases from the core promoter. By binding different regulatory proteins, help (binds activators) or hinder (binds repressors) transcription from the core promoter.

29 3.2 RNA Pol II forms a pre-initiation complex with GTFs at the promoter TBP in TFIID binds to the TATA box TFIIA and TFIIB are recruited with TFIIB binding to the BRE RNA Pol II-TFIIF complex is then recruited TFIIE and TFIIH then bind upstream of Pol II to form the pre-initiation complex Promoter melting using energy from ATP hydrolysis by TFIIH

30 3.3 Promoter escape requires phosphorylation of the polymerase Tail Promoter escapes after the phosphorylation of the CTD tail tail : CTD (carboxy-terminal domain) of pol II Repeats of (Tyr-Ser-Pro-Thr-Ser-Pro-Ser).

31 3.4 Transcription initiation in vivo requires additional proteins Assembly of the pre-initiation complex in presence of Mediator, histone modifiers and nucleosome remodelers, and transcription activators Mediator

32 3.5 A new set of factors stimulate Pol II elongation and RNA proofreading Once polymerase has escaped the promoter and initiated transcription, it shifts into the elongation phase. This transition involves the Pol II enzyme shedding most of its initiation factors for example, the general transcription factors and Mediator. In their place, another set of factors is recruited. Some of these are elongation factors. Others are required for RNA processing. They are recruited to the CTD tail of the large subunit of Pol II. Phosphorylation of the CTD tail leads to an exchange of initiation factor for those factors required for elongation and RNA processing.

33 P-TEFb (a kinase stimulating elongation in 3 separate ways): Phosphorylates the serine residue at position 2 of the CTD repeats Activates hspt5, an elongator Recruits another elongator, TAT-SF1 TFIIS: Stimulates the overall rate of elongation by limiting the time that polymerase pauses; Contributes to proofreading by stimulating an inherent RNase activity in Pol II (hydrolytic editing in prokaryotes)

34 3.6 Elongating polymerase must deal with histones in its path FACT--facilitates chromatin transcription; a heterodimer of two conserved proteins: Spt16 (binds H2A/H2B dimer ) and SSRP1 (binds H3/H4 tetramer )

35 3.7 RNA processing enzymes are recruited by the CTD tail of polymerase

36 The structure and formation of the 5 RNA cap

37 3 polyadenylation CPSF--cleavage and polyadenylation specificity factor; CstF--cleavage stimulation factor

38 3.8 Models of termination: Torpedo and allosteric

4. Transcription by RNA polymerase I and III RNA Pol I & III recognize distinct promoters, using distinct sets of transcription factors, but still require TBP; The

39 4. Transcription by RNA polymerase I and III RNA Pol I & III recognize distinct promoters, using distinct sets of transcription factors, but still require TBP; The promoter for the rrna gene comprises a core element and a UCE (upstream control element) Accordingly, two factors, SL1 and UBF, are required for initiation besides Pol I.

40 Most Pol III promoters are found downstream of transcription start site; consisting BoxA and BoxB for trna genes or A and C for 5S rrna genes; TF III B and C are required for trna initiation, while TF III A plus are for 5S rrna

DNA Transcription. Dr Aliwaini

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