1. LECTURE CONNECTIONS 13 | Transcription © 2009 W. H. Freeman and Company

2. Transcription Initiation: the transcription apparatus assembles on the promoter and begins synthesis of RNA; Elongation: DNA is threaded through RNA polymerase, the polymerase unwinding the DNA and adding new nucleotides, one at a time, to the _____ end of the growing RNA strand; Termination: the recognition of the end of the transcription unit and the separation of the RNA molecule from the DNA template.

3. Initiation The transcription apparatus: Bacterial RNA polymerase: five subunits made up of the core enzyme. The sigma  factor: binding to the promoter when transcription starts. The substrate for transcription: Ribonucleoside triphosphates – rNTPs added to the 3′ end of the RNA molecule

4. Initiation Bacterial promoters: Consensus sequences: sequences that possess considerable similarity −10 consensus: 10 bp upstream of the start site Pribnow box: 5′ TATAAT 3′ 3′ ATATTA 5′ −35 consensus sequence: TTGACA

7. Concept Check 2 What binds to the −10 consensus sequence found in most bacterial promoters? a.The holoenzyme (core enzyme + sigma factor) b.The sigma factor alone c.The core enzyme alone d.mRNA

8. Initiation Initial RNA synthesis: No primer is required. The location of the consensus sequence determines the position of the start site. At the end of initiation, RNA polymerase undergoes a change in conformation and no longer can bind to the consensus sequences in the promoter.

9. Elongation RNA elongation is carried out by the action of RNA polymerase. The sigma subunit is usually released after initiation. RNA polymerase progressively unwinds the DNA at the leading edge of transcription bubble and rewind the DNA at the upstream edge of the bubble. RNA polymerase is capable of a type of proofreading during transcription (it can back up and cleave nucleotides)

10. Termination Transcription stops after the terminator has been transcribed. Events necessary to stop transcription: - RNA polymerase must stop synthesizing RNA - RNA molecule must be released from RNA polymerase - The newly made RNA molecule must dissociate fully from the DNA - RNA polymerase must detach from the DNA template Bacterial cells possess 2 major types of terminators. - Rho- independent terminator - Rho- dependent terminator (require an ancillary protein called Rho)

11. Rho-independent terminator: -Contain inverted repeats - a string of approximately 6 adenine nucleotides follows the second inverted repeat in the template DNA. hairpin structure formed by inverted repeats, followed by a string of uracils

13. Rho-dependent terminator: - DNA sequences that produce a pause in transcription; - a DNA sequence that encodes a stretch of RNA upstream of the terminator that is devoid of any secondary structures.

14. 13.4 The Process of Eukaryotic Transcription Is Similar to Bacterial Transcription but Has Some Important Differences In eukaryotes, there are 3 different RNA polymerases, each of which transcribes a different class of RNA and recognizes a different type of promoter; A promoter’s description depends on whether the promoter is recognized by RNA polymerase I, II or III; Different kind of promoters require different kind of proteins,

15. Transcription and Nucleosome Structure – Chromatin modification before transcription Promoters: Basal transcription apparatus (general transcription factors + RNA polymerase) Transcriptional activator proteins (bind to specific DNA sequences and bring about higher levels of transcription) RNA polymerase II – mRNA synthesis

16. Transcription and Nucleosome Structure – Chromatin modification before transcription Promoters: Core promoter TATA box (TATAAAA) −25 to −30 bp, binded by transcription factors Regulatory promoter A variety of different consensus sequences may be found in the regulatory promoters.

18. Transcription and Nucleosome Structure – Chromatin modification before transcription Enhancers: sequence that stimulates maximal transcription of distant genes in the same DNA molecule Polymerase I and polymerase III promoters recognize promoters that are distinct from those recognized by RNA polymerase II. Example: promoters for small rRNA and tRNA genes

19. Initiation Transcription is initiated through the assembly of the transcriptional machinery on the promoter. RNA polymerase II + transcription factors Basal transcription factors: TFIID- subunit TBP (TATA binding protein) TFIIA- stabilizes TFIID TFIIE and TFIIF: release the RNA polymerase from the complex and participate on the unwinding of the DNA

21. Elongation Many of the transcription factors are left behind at the promoter and can serve to quickly reinitiate transcription with another RNA polymerase enzyme. The RNA polymerase maintains a transcriptional bubble during elongation.

22. Termination RNA polymerase I: requires a terminator factor that binds to a DNA sequence downstream of the termination site. RNA polymerase II: ends transcription after transcribing a terminator sequence that produces a string of uracil nucleotides in the RNA molecule. RNA polymerase III: does not require that a hairpin structure precede the string of Us.

23. Concept Check 3 What is the difference between the core promoter and the regulatory promoter? a.Only the core promoter has consensus sequences. b.The regulatory promoter is farther upstream from the gene. c.Transcription factors bind to the core promoter; transcriptional activator proteins bind to the regulatory promoters. d.Both b and c above