1. Chapter 13 - Transcription

2. RNA structure Nucleotides Ribose sugar – OH at 2′ C Nitrogenous bases
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 Secondary 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) Messenger RNA (mRNA)
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 RNA coding region Terminator
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