lec 1 Flashcards
Chromatin condensation
inactivates gene transcription:
Blocks RNA polymerases and general transcription factors from interacting with gene promoters
Repressor proteins
- May bind to transcription-control elements to inhibit transcription initiation by Pol II
- May interact with multiprotein co-repressor complexes to condense chromatin
Pioneer transcription factor
- Binds to a specific regulatory sequence within the condensed chromatin
- Interacts with chromatin-remodeling enzymes and histone acetylases that decondense the chromatin, making it accessible to RNA polymerase II and general transcription factors
Activator proteins:
- Bind to specific transcription-control elements in both promoter-proximal sites and distant enhancers
- Interact with one another and with the multisubunit Mediator complex to assemble general transcription factors and RNA Pol II on promoters
Transcriptional activation
- Pol II initiates transcription
- Pol II pauses after transcribing fewer than 100 nucleotides because of action of the elongation inhibitor NELF (negative elongation factor) associated with DSIF (DRB sensitivity-inducing factor)
Activators
- They promote association of the Pol II-NELF-DSIF complex with elongation factor P-TEFb [cyclin T-CDK9 (kinase)], which releases NELF
- NELF release allows resumption of RNA transcription
Differences between prokaryotes and eukaryotes
- Transcription in eukaryotes takes place on DNA that is wrapped in chromatin
- Chromatin needs to open for a gene to be activated and transcription to proceed
- Chromatin-mediated regulation is often called EPIGENETIC REGULATION of gene expression
Heterochromatin
- regions of chromosomes that are intensely stained
- DNA is more densely packed
- rich in repetitive DNA (Transposons, centromeres and telomeres)
- not accessible to transcriptional machinery
- Inactive genes are found in heterochromatin
Euchromatin
- lightly stained chromosome regions
- Active genes are found in euchromatin
- accessible to transcriptional machinery
RNA polymerase I
Pre-rRNA (Ribosome components, protein synthesis)
RNA polymerase II
- mRNA (encodes protein)
- snRNA (RNA splicing)
- siRNA (chromatin-mediated repression & translation control)
- miRNA (translation control)
RNA polymerase III
- tRNA (protein synthesis)
- 5S rRNA (ribosome components & protein synthesis)
- snRNA U6 (RNA splicing)
- 7S RNA (insertion of polypeptides into ER)
- other small stable RNA (various functions)
Similarities between the different RNA pol (eukaryotic)
- RNA pol II consists of 12 polypeptides, called RPB1, RPB2…RPB12
- All other eukaryotic RNA polymerases share very high level of homology with the yeast RNA pol II
CLAMP DOMAIN
- in the polymerase (RPB1) accommodates DNA
- After positioning over DNA the clamp is closed by a bridge
catalytic center
- where the synthesis of RNA takes place with the participation of Mg++
- The synthesized RNA exits through a ”channel” and is immediately capped by 7m Guanosine.
Eukaryotic RNA pol structure
- Eukaryotic RNA polymerases contain two large subunits and 10–14 smaller subunits, some of which are common between two or all three of the polymerases, whereas others are specific.
eukaryotic and prokaryotic pol II similarities
RPB1 & RPB2 = B’ & B
CTD
- Eukaryotic RNA polymerase II contains a unique Carboxy-Terminal Domain (CTD) of its RPB1 subunit
- specialized domain not found in any other polymerase, prokaryotic or eukaryotic
- involved in multiple regulatory interactions and plays a key role in the initiation, release, elongation and processing of the synthesized mRNAs
- The CTD in yeast contains of 26 repeats of Tyr-Ser-Pro-Thr-Ser-Pro-Ser (in mammals it contains 52 repeats)
- The Ser residues in the CTD are phosphorylated upon transition from initiation to elongation
- Nonphosphorylated CTD initiates transcription
- Phosphorylated CTD as polymerase initiates transcription and moves away from the promoter; many of the serine and some tyrosine residues in the CTD become phosphorylated.
The RNA pol II – transcribed genes are regulated by:
- Conserved basal promoter elements aka CORE PROMOTER SEQUENCES
- Promoter-proximal binding sites for transcriptional activators
- Distal enhancers or repressors
- Chromatin structure
Core Promoter Elements
Specify where polymerase initiates transcription of an RNA complementary to the template strand of the gene DNA.
- BRE (TFIIB Recognition Element)
- TATA box
- Initiator
- DPE (Downstream Promoter Element)
- CpG islands
BRE (TFIIB recognition element)
- influence the activity of the promoter
TATA box
- a tight consensus sequence; prevalent in highly transcribed genes
- single base change decreases gene transcription.
Initiator
- degenerate sequence
- less conserved element
- some genes contain Initiator but no TATA
DPE (Downstream promoter element)
- influence the activity of the promoter
CpG islands
- “p” represents the phosphate between the C and G nucleotides
- Control housekeeping genes expressed at low constant levels.
- Harder to bend around nucleosomes — form nucleosome-free regions.
RNA polymerases must recognize the promoter and correctly INITIATE transcription at a very specific position
They can not do this alone!!!
GENERAL POLII TRASCRIPTION FACTORS (GTFs) assemble the so called preinitiation complex over the CORE PROMOTER SEQUENCES
- Other factors (DNA helicase) help the polymerase initiate transcription
- Other factors (protein kinase) release the polymerase
- Other factors help the polymerase elongate
- Other factors move nucleosomes out of the way
RNA Pol I GTFS:
- TFI
- TFIA
- TFIB
RNA Pol II GTFs
- TFIIA
- TFIIB
- TFIID
- TFIIE
- TFIIH
RNA Pol III GTFs:
- TFIII
- TFIIIB
- TFIIIS
