Chapter 13 - Transcription

1. Chapter 13 - Transcription

2. RNA structure • Nucleotides • Ribose sugar – OH at 2′ C • Unstable; short-lived molecule • Nitrogenous bases • Adenine • Guanine • Cytosine • Uracil

3. RNA structure • Nucleotide polymer held together by phosphodiester bonds • Usually single-stranded • Due to short regions of complementary sequences, can base pair to form stems, hairpins, etc

4. RNA structure • Primary structure • Nucleotide sequence • Secondary structure • Formed by complementary regions • Has greater variety than helix of DNA • Various shapes have different functions

5. Classes of RNA • Ribosomal RNA (rRNA) • Joins with protein subunits to form ribosomes • Site of polypeptide synthesis • Messenger RNA (mRNA) • Codes for a polypeptide • Amino acid sequence • Pre-messenger/primary transcript • In eukaryotic cells only • Needs to be modified before exiting the nucleus • Prokaryotic mRNA can start to be translated before transcription is complete • Transfer RNA (tRNA) • Brings specific amino acid to the ribosome for incorporation into the growing polypeptide

6. Classes of RNA cont • Small nuclear RNA (snRNA) • Joins with small nuclear proteins to form snRNPs – small nuclear ribonuclear proteins • Assist with post-transcriptional modifications of primary transcript • Splices out introns • Small nucleolar RNA (snoRNA) • Aids in the processing of rRNA

7. Classes of RNA cont • MicroRNA (miRNA) and small interfering RNA (siRNA) • In eukaryotic cells • RNAi – RNA interference • Initiates degradation or inhibition of mRNA molecules • Piwi-interacting RNA (piRNA) • Found in mammalian testes • Regulation of sperm development

8. Synthesizing RNA from DNA • During DNA replication, the entire DNA molecule is copied • In transcription, only a small section of DNA is used for the synthesis of RNA • Usually one gene at a time, or several genes (in prokaryotes) • Only one of the two strands of DNA gets transcribed into RNA • Transcribed/template strand • Nontemplate strand = coding strand • “coding” strand gives RNA sequence (replace T with U)

10. Synthesizing RNA from DNA cont • In DNA, one strand may be the template strand for one gene, while another strand may be the template strand for another gene • Transcription occurs in the 5′→3′ direction of the RNA molecule • Complementary and antiparallel to the DNA strand

11. Transcription Unit • Promotor • Upstream from coding region • Specific DNA sequence • Serves as attachment site for transcription molecules • Sequence is NOT transcribed into RNA • RNA coding region • Terminator • Downstream from coding region • Is transcribed into RNA; sequence is later removed • Specific sequence to halt transcription

12. RNA polymerase • Does NOT require a primer • Prokaryotic RNA polymerase • Single type of polymerase used for all transcription • Composed of 5 polypeptide subunits – core enzyme • (σ) sigma factor • Binds with core enzyme to create holoenzyme • Controls binding to promotor • Without sigma, polymerase will bind anywhere on DNA • Various sigma factors are present for different promotor types • Releases from core protein after transcript is several nucleotides long • Eukaryotic RNA polymerase • Different classes for different types of RNA • Consists of multiple subunits • Core enzyme with accessory proteins at different stages

14. Bacterial transcription • Initiation • Specific DNA sequence at promotor • Consensus sequence • Most common nucleotides in a particular position • R = purine • Y = pyrimidine • N = any

15. Promotor • Two consensus sequences • Any change/mutation in promotor region alters the rate of transcription • Down mutation – reduces rate of transcription • Up mutation – increases rate of transcription • rare

16. Holoenzyme • Binds to promotor consensus sequences only, but enzyme covers larger area • Polymerase alters its structure and binds more tightly, unwinding DNA • Begins at -10 sequence and continues downstream • Bases on consensus sequence location, enzyme’s active site is in position +1 • First RNA nucleotide is placed complementary to DNA sequence

17. Elongation • After transcript is approximately 12 nucleotides long, polymerase structure alters so it is no longer bound to consensus sequences • Moves downstream • Sigma factor is usually released • Polymerase continues to unwind DNA downstream and rewind upstream • Transcription bubble • Positive supercoiling ahead of bubble; negative supercoiling behind • Topoisomerase enzymes relieve tension

18. Termination – Rho-independent • Contains inverted complementary sequences that form a hairpin when transcribed • Slows transciption • 2nd repeat sequence is polyA (polyU on RNA) • Weak (due to 2 H bonds between each), and transcript separates from DNA template

19. Termination – Rho-dependent • Rho factor protein • Binds to regions with no secondary structure • RNA sequence upstream from termination doesn’t form secondary structure • Rho factor binds to RNA and moves toward 3′ end • At a hairpin, transcription slows and rho factor can “catch up” to DNA/RNA • Rho has helicase activity • Breaks H bonds and separates RNA from DNA

20. Modifications for eukaryotic transcription • Nucleosome structure • DNA associated with histone proteins • Acetylation of histones reduces their positive charge; makes DNA more accessible • Initiators • Promotors • Have varied sequences to attract different polymerase types • Polymerases have several associated accessory proteins • Directly upstream from gene • Enhancers • Can be located far away from gene • DNA loops around to bring enhancer (with activator protein) to promotor region • Some sequences can be repressors/silencers • Termination • Different polymerases have different mechanisms for termination