Transcription and Gene Expression Flashcards
RNA Pol polymerizes RNA in what direction? Are primers required?
RNA Pol moves in 5’-3’ direction
primers NOT required
but DNA strand serves as template (DNA-dependent RNA polymerase)
higher error rate than DNA polymerase, but this is OK because proteins are not inherited as genes are and mutated protein is less consequential than mutated gene
what kind of reaction does RNA pol catalyze? (organic chemistry)
nucleophilic attack to break alpha and beta phosphate on innermost phosphate group to create phosphodiester bond
what do RNA pol I, II, and III do in humans
RNA pol I —> rRNA synthesis
RNA pol II —> transcription (DNA —> mRNA)
RNA pol III —> tRNA synthesis
TATA box
DNA sequence present in promoters, which control initiation of RNA transcription
part of core (basal) promoter
cis-regulatory element
contrast cis-regulatory and trans-regulatory elements
cis-regulatory elements: present on same molecule of DNA that they regulate, make up core (basal) promoter clustered around the +1 base
trans-regulatory elements: proteins that regulate gene expression, freely diffuse within the cell
how is the RNA Pol II pre-initiation complex (PIC) assembled
General Transcription Factors (GTFs, aka basal transcription factors) bind to core promoter, recruit RNA Pol II
- TATA binding protein (TBP) recognizes TATA box
- PIC assembles
- RNA Pol II recruited by GTFs bound to core promoter
- RNA Pol II binding triggers more GTFs activity and helicase activity to unwind DNA (transcription bubble forms)
how does the C-terminal domain (CTD) of RNA pol II contribute to promoter escape in transcription?
CTD contains peptide repeats rich in Tyr, Ser, Thr -> contain hydroxyl groups that can be phosphorylated
kinase phosphorylate CTD tail, triggering promoter escape
different phosphorylation patterns of CTD serve as scaffolds for being in to different proteins that act on the newly transcribed mRNA
Which of these statements is true:
a. RNA polymerase molecules that are farther away from promoter region will have shorter RNA transcripts associated with the transcription bubble
b. promoters have directionality
TRUE: promoters have directionality
RNA pol farther away from promoter region will be associated with longer RNA transcripts (they’ve made more progress)
group these together:
template strand
positive strand
non-coding strand
sense strand
antisense strand
non-template strand
coding strand
negative strand
template strand = non-coding = negative strand = antisense strand
non-template strand = coding strand = positive strand = sense strand
transcribed mRNA will have the same sequence as the ____ strand but with uracil instead of thymine (give all 4 names)
coding strand / non-template strand / positive strand / sense strand
Your research team discovers a single-strand, positive sense RNA virus. If this virus were released into human cytoplasm, could it be directly translated by human ribosomes to produce viral proteins?
yes, the positive/coding/sense strand contains the code needed to produce the viral proteins, and because it is an RNA virus it already has the uracil needed for translation
Your clinical research team is hoping to develop a viral treatment against tumors. You deliver a single-stranded positive strand RNA genome into the cancer cells in order to produce viral RNA that will cause cell death, using human RNA polymerases. Why won’t this work?
eukaryotic polymerases need DNA template to make more RNA
viruses are packaged with their own RNA-dependent RNA polymerase
how does Molnupiravir cause viral mutagenesis in SARS-CoV-2?
substrate for RNA-dependent RNA polymerase
drug is incorporated into viral RNA and causes mutagenesis
Molnupiravir is a prodrug
which of these statements is true of prokaryotic transcription:
a. prokaryotic genes contain introns which are spliced from the pre-mRNA
b. transcription is performed by RNA Pol II
c. the ATG codon is the translation start site
the ATG codon is the translation start site in prokaryotic cells
prokaryotic genes do NOT have introns
a SINGLE RNA Pol performs all transcription in prokaryotes (do not confuse with multiple DNA Pol in prokaryotes)
instead of general transcription factors, promoter sequences in prokaryotes are recognized by…
sigma factors, which recruit RNA pol
during promoter escape, sigma factors remain bound to promoter with only RNA pol transitioning to elongation phase
different sigma factors recognize different promoter sequences
bacterial transcription termination comes in 2 flavors (hint: dependent vs not). Describe the differences
Rho-dependent: Rho acts as RNA-DNA helicase, hydrolyzes ATP to unwind
Rho-independent: termination sequence within DNA template facilitates stable hairpin loop and string of “U” residues (that will bond with A, weaker than G to C bond) to facilitate separation
how does the antibiotic Rifampicin (aka rifampin) work?
specifically inhibits RNA pol without affecting human RNA pols
used in combination therapy to treat M. tuberculosis
what are the 3 pieces of mRNA processing (eukaryotes)
5’ capping - stabilizes mRNA, important for export of RNA form nucleus to cytoplasm
splicing
3’ polyadenylation - stabilizes mRNA and important for export also
where does splicing occur within the cell?
nucleus
5’ splice site (splice donor, GU), branch site, and 3’ splice site (splice acceptor, AG)
requires spliceosomes made of snRNAs and proteins (= snRNPs)
forms lariat (loop), with the branch point adenine having an unusual 2’-5’ phosphodiester bond in addition
beta thalassemia is what kind of mutation
intron mutation
what role does the polyadenylation signal sequence play in addition of a poly A tail?
polyadenylation signal sequence is a marker - 10-30bp downstream the sequence is cleaved
poly A tail is added after sequence is cleaved
[adenines come form ATP, not a template - energy input required]
what processing happens to get pre-mRNA and mature mRNA
remove promoter/transcription factors —> pre-mRNA
remove introns, add modifications —> mature mRNA
T/F: eukaryotic mRNA processing is coupled to transcription
TRUE: modifications occur during transcription (requires C-terminal domain/CTD of RNA Pol II —> creates phosphorylation patterns)
[in prokaryotes, transcription and translation are coupled]
a mature mRNA has 1000 nucleotides. The 5’ UTR is 50 nucleotides, and the 3’ UTR is 50 nucleotides.
How many amino acids are in the protein this mRNA encodes?
1000 - (50 + 50) = 900
900 / 3 = 300 amino acids
[there are 3 base pairs per codon]
name 3 grouped methods of regulating the rate of transcription
- promoters, enhancers, silencers
- chromatin remodeling
- DNA methylation and histone modifications (epigenetics)
what is the purpose of alternative splicing
production of multiple proteins by a single gene
what metabolic pathway is PEPCK a key enzyme in, and how is its expression regulated (2 answers)?
PEPCK = phosphoenol pyruvate carboxykinase, enzyme in gluconeogenesis (liver)
- glucagon (peptide) binds receptor —> activates promoters with CRE (cAMP response element)
- cortisol (steroid) diffuses into cell —> activates promoters with GRE (glucocorticoid response element)
how does glucagon increase transcription of PEPCK?
walk through the steps in the pathway
- glucagon (peptide) binds receptor, triggers conformational change that activates adenylyl cyclase
- AC catalyzes cAMP formation, which activates PKA
- PKA phosphorylates CRE binding protein (transcription factor) in the nucleus
- CREB(p) binds CRE (cAMP response element) DNA sequence upstream of PEPCK gene
- PEPCK gene transcription turned on
how does cortisol increase PEPCK transcription?
walk through the steps
- cortisol (steroid) diffuses into cell and binds receptor in cytosol, causing conformational change that exposes nuclear localization signal
- complex moves to nucleus and binds DNA sequence GRE (Glucocorticoid response element) upstream of PEPCK gene
- PEPCK transcription turns on
what is thalassemia and its cause
single gene disorder associated with reduced hemoglobin synthesis
due to loss or reduced amounts of either alpha or beta subunits of hemoglobin (2 alpha, 2 beta subunits to make tetrameter)
protein loss denoted as alpha/beta^0
reduced protein denoted as alpha/beta^+
how is hemoglobin transitioned from fetal (HbF, gamma subunits instead of beta) to adult (HbA, alpha and beta)?
locus control region (LCR) turns on gamma globulin in utero
after birth repressor protein (BCL11A) binds regulatory sequence and suppresses gamma-chain expression
LCR turns on beta globulin
[hereditary persistence of fetal hemoglobin - asymptomatic, HbF continues to be made in adulthood]
why should you not eat a mushroom that contains alpha-amanitin?
alpha-amanitin inhibits the activity of RNA Pol II —> halts transcription
[also affects Pol III, but not Pol I]
Hutchinson-Gifford Progeria is caused by a C>T SNP that changes codon GGC > GGT. Both codons encode for Gly (glycine). What could explain this?
this SNP could create an alternative splice site in the gene
which is true of RNA Pol II:
a. transcribes both strands, one moving 3’>5’ and the other 5’>3’
b. only one DNA strand, moving 3’>5’
RNA Pol II transcribes only TEMPLATE strand, 3’>5’
this way, growing RNA chain is 5’>3’
during what process would you observe the formation of an RNA species with an adenine containing three phosphodiester bonds, linked to the 2’, 3’, and 5’ carbons?
splicing of hnRNA (heteronuclear RNA, collective term for unprocessed/pre-mRNA)
introns are spliced out of mRNA as lariat molecules (transient structure)
which of these is a feature of transcription but NOT replication?
a. transcription is primer-dependent
b. RNA strand is elongated in 3’>5’ direction
c. transcription is template independent
d. RNA strand is elongated in 5’>3’ direction
transcription is primer INDEPENDENT - RNA Pol can start from “scratch”
RNA is elongated 5’>3’ in both replication and transcription (never 3’>5’)
transcription is dependent on a template (so is replication)
which of these can enhancers mediate:
a. recruit ATP-dependent chromatin-remodeling enzymes
b. recruits histone acetyltransferases
c. recruits basal transcription factors
all of above: enhancers can
- recruit ATP-dependent chromatin-remodeling enzymes to increase accessibility for transcription machinery, including RNA pol II
- recruit histone acetyltransferases to unpack nucleosomes
- recruit basal transcription factors to core promoter
contrast transcription termination in prokaryotes and eukaryotes
prokaryotes: rho-dependent or rho-independent (hairpin loop followed by A-U base pairing)
eukaryotes: polyA signal triggers endonucleolytic cleavage downstream and addition of polyA tail
The glucocorticoid receptor protein contains a Zinc Finger domain that is important for interaction with GRE. If a mutation affected the receptor protein ability to bind zinc, which of these would be most directly affected?
a. ligand binding
b. transcriptional activation
c. nuclear localization
d. DNA binding
DNA binding - GRE is a DNA element (this would be most directly affected, followed by transcriptional activation after that)
these are all the first steps of a process. name the process for each:
a. binding of GTFs and RNA pol to a promoter sequence
b. binding of tRNA to IF2
c. binding of DnaA to the origin
a. binding of GTFs and RNA pol to promoter sequence —> first step of eukaryotic initiation of transcription
b. binding of tRNA to IF2 —> first step of translation
c. binding of DnaA to origin —> first step of prokaryotic DNA replication
Rifampicin is a drug active against what enzyme?
bacterial RNA pol
Rifampicin is an antibiotic
