Transcription, RNA Processing, and Regulation of Gene Expression Flashcards
DNA Topoisomerases
Essential for replication; not necessarily essential for transcription
DNA Helicases
the “zippers” that separate the two strands of DNA; ESSENTIAL for transcription
- RNAP; TFIIH
RNA Polymerase (prokaryotic)
Transcribes all RNA
RNA Pol I
- transcribes: rRNA
- location: nucleolus
- function: ribosome assembly
RNA Pol II
- transcribes: hnRNA/mRNA
- location: nucleus
- function: protein synthesis
RNA Pol III
- transcribes: tRNA, miRNA, siRNA, snRNA, snoRNA
- location: nucleus
- function: protein synthesis (cytoplasm); diverse nuclear functions
RNA polymerase (mitochondrial)
- transcribes: all mitochondrial RNAs
- location: mitochondria
- function: transcription of mitochondrial genes
Prokaryotic (bacterial) RNA Polymerase
Core: alpha x2, beta x2, omega
Holo: includes sigma
- sigma confers promoter recognition to RNA pol
- sigma is not required for catalytic activity of RNA pol
3 Stages of Transcription
- Initiation: RNAP/Pol II binds promoter (sigma recognizes promoter in prok); no primer required; principal site for REGULATION of transcription
- Elongation: lengthens transcript; Holo-RNAP dissociates from sigma subunit (prok); RNAP II dissociates from GTF (euk)
- Termination: RNA synthesis stops; depends on DNA encoded RNA signals (DNA says when, RNA does the stopping)
Genetic Elements of Bacterial Operon
- structural genes
- operon
- promoter
- activator binding site
- operator
- concept
- structural genes: encode protein; down stream of all
- operon: mRNA encoded info; transcribed as single mRNA chain
- promoter: binding site for RNA polymerase
- activator binding site: DNA sequence that binds a protein that enhances transcription
- operator: DNA sequence that binds proteins that inhibits transcription; blocks holoenzyme
- concept: compact arrangement of DNA sequence elements: often less than 150bp of DNA from start site of transcription to activator binding site
Structure of Bacterial Promoter
-35 sequence — 19 base pairs — -10 Pribnow Box (TATAAT) — ~ 2 bp — Start of transcription
Rho-dependent Transcription Termination
PROKARYOTES ONLY
- termination signal is encoded in the gene
- signal is manifest as RNA
- results in dissociation of the DNA-RNA-protein-complex
Rho = torpedo
RNA pol = ship
Termination = BOOM
Rho-independent Transcription Termination
PROKARYOTES ONLY
Stem-Loop (S-L)
- RNA S-L structure is encoded in DNA and functional as RNA
- structural features: S-L and a run of UUUUUU immediately following
- terminates transcription within the catalytic core of RNAP (in CONTRAST with RHO-dep)
Rifampicin Antibiotic
binds RNA polymerase and inhibits initiation of transcription
RNA polymerase II
EUKARYOTES
- 12 subunits; large complex with more subunits than prok; some subunits look and act similar to prok
- NO SIGMA COUNTERPART IN RNAP II
Eukaryotic Transcription (Pol II) Core Promoter
REGULATORY dsDNA sequence
- transcription start site (INR): +1
- TATA box: -25
- DPE (downstream promoter element): +30
- promoter function: binding site for RNA Pol II and “GTF”s
Eukaryotic Transcription (Pol II) Proximal Promoter
REGULATORY ds DNA sequence; NOT A BINDING SITE FOR RNA POL II
- <120 bp from start
- binding site for activator proteins
Eukaryotic Transcription (Pol II) Enhancer
REGULATORY dsDNA sequence; binds regulatory proteins
- > 250 bp from start (far from genes controlling)
- function in either orientation
- can act anywhere
- binding site for transcription activators
General Transcription Factors
multi sub unit GTF that has responsibility to recognize promoter DNA sequences and tell RNA poly where to associate with DNA
*** TFIID ALONE NOT sufficient to create RNA pol binding site
TFIID
Equivalent of sigma factor
* recognizes promoter*
ALONE NOT SUFFICIENT FOR BINDING
GTF assembly
assemble on promoter in a stepwise manner
Pol II Binding orientation
nothin on Pol II recognizes the DNA; doesnt have to because Pol II DOESNT touch DNA, instead touches TF
TFIIH
- involved in transcription coupled repair (NER)
- required to melt promoter DNA
5’ Cap
- not encoded in DNA
- protects 5’ end from exonucleases
- important in translation initiation: bound by eIF4E
eIF4E
binds 5’ cap and initiates protein synthesis in cytoplasm
PolyA tail
- encoded in DNA
- recognized and function in RNA
- endonuclease recognizes and cuts at the end; polyA tail added
Transcription-coupled DNA repair
-NER pathway
- fast, preferential repair of the transcribed strand of an active gene
TCR factors include subunits of TFIIH, including two helicases, XPB and XPD, proteins whose genes products are known to be involved in the disease Xeroderma pigmenosum (XP), Tricothiodystropy (TTD) and Cockayne Syndrome (CS)
Transcription-coupled repair
sub-pathway of NER that repairs the TRANSCRIBED DNA strand faster than the rest of the genome
***TCR requires TFIIH subunits XPB and XPD)
Cockayne Syndrome
premature aging syndrome with developmental and neurological defects
a-amanitin?
Alpha-amanitin
binds to RNAP II and inhibits the elongation stage of transcription (starts but can’t be completed)
Define:
5’ splice site
3’ splice site
5’ splice site: splice donor site
3’ splice site: splice acceptor site
Intron starts/ends
starts 5’ GU ends 3’ AG
INTRON TELLS WHERE TO SPLICE
RNA Chain Phosphodiester Bonds
3’ (base n) to 5’ (base n+1)
ALL nucleotides in RNA contain a 2’-OH group
SNURPs
small, nuclear, uracil rich, RNA-protein complexes
Spliceosome
large complex that removes introns from pre-RNA
U1
SNURP
guide sequence; recognizes invariant sequence at 5’ splice site
U2
SNURP
recognizes branch site (special A; use it’s OH)
U1&2
recognize each other and conformationally change to promote A-OH linkage and splicing
- conformational change requires ATP
- splicing does not require ATP
B-thalassemia
results from intronic mutation
- mutation in DNA but effect seen on RNA splicing level
- splice sites changed by point mutations which can incorrectly start/stop intron splicing at incorrect locations
Alternative Splicing
a gene produces multiple proteins from the same DNA sequence
UTR and Introns
Introns can be found in UTR- REGULATION
Upstream- “leader region”; regulatory effects; change in protein expression
Downstream- change in stability; regulatory effects
Transcriptional Activators
hormones BOUND to receptor proteins interacting with enhancer sequences
Agonist
substance which initiates a physiological response when combined with a receptor
- bind hormone receptors
- stimulate receptor activity; stimulate gene expression
I.e. anabolic steroids; fentanyl
Antagonist
substance that interferes with or inhibits the physicological action of another
- bind hormone receptors
- block receptor acrtivity; repress gene expression
i. e. tamoxifen; naloxone
How to answer test Qs on introns/exons
exon 1 | intron 1 | exon 2
- where’s mutation
- draw out new mRNA and then answer question
