Exam 3 Lectures Flashcards
what is transcription
DNA to RNA
what is translation
RNA to proteins
How many strands of RNA
single stranded
RNA contains what sugar and what base
ribose, uracil
RNA can for hydrogen bonds with what
DNA
RNA can form what with complementary sequences of the strand
conventional base pairs and also non conventional base pairs
what are the non conventional bp
GU, AG, CU
Which of the following correctly describes the differences between RNA and DNA?
A) RNA contains the sugar deoxyribose, whereas DNA contains ribose.
B) RNA uses the base uracil, which differs from thymine in DNA by the presence of a C=O group.
C) RNA contains ribose, which differs from deoxyribose in DNA by the presence of an additional –
OH group.
D) RNA uses the base thymine, which differs from uracil in DNA by the presence of a –CH3 group.
E) RNA contains deoxyribose, and DNA contains ribose.
C
transcription produces RNA complimentary to what
one strand of DNA
what carries out DNA transcription
RNA polymerase
enzyme that transcribes DNA into RNA, moves stepwise along DNA unwinding helix at its active site
RNA polymerase
how does RNA polymerase move
stepwise along DNA
catalysis for RNA polymerase
Mg2+
energy source of RNA polymerase
uses ribonucleoside triphosphate for polymerization, powered by phosphate bond energy
what are the ribonucleoside triphoshates
atp, utp, ctp, gtp
messenger RNAs, code for proteins
mRNAs
, form basic structure of ribosome and catalyze protein synthesis
rRNAs
central to protein synthesis as the adaptors between mRNA and amino acids
tRNAs
serves as the template for the telomerase enzyme that extends the ends of chromosomes
telomerase RNA
function in a variety of nuclear processes including the splicing of pre mRNA
snRNAs
help to process and chemically modify rRNAs
snoRNAs
, not all of which appear to have a function, some serve as scaffolds and regulate diverse cell processes, including X chromosome inactivation
lncRNAs
regulate gene expression by blocking translation of specific mRNAs and causing their degradation
miRNAs
, turn off gene expression by direction the degradation of selective mRNAs and helping to establish repressive chromatin structures
siRNAs
bind to piwi proteins and protect the germ line from transposable elements
piRNAs
what genes does RNA poly 1 transcribe
5.8S, 18S, and 28 S rRNA genes
RNA poly 2 transcribes what genes
all protein coding genes, plus snoRNA genes, miRNA genes, siRNA genes, lncRNA genes, and most snRNA genes
RNA poly 3 transcribes what genes
tRNA genes, 5S rRNA genes, some snRNA genes, and genes for other small RNAs
rRNAs named according to what which refer to what
S values, refer to rate of sedimentation in an ultracentrifuge, larger S value the larger the rRNA
Which of the following accurately describes the process of transcription by RNA polymerase?
A) RNA polymerase adds nucleotides to the DNA strand during transcription.
B) The RNA transcript is a complementary copy of both DNA strands.
C) RNA polymerase moves along DNA, unwinding the helix at its active site, with the help of Mg²⁺.
D) The RNA polymerase forms a long, stable DNA–RNA helix that remains intact during
transcription.
E) Ribonucleoside triphosphates are not involved in the energy process during transcription.
C
in euk mRNA processing, what is included in it
contains both exons and introns
what happens at both ends of euk mRNA
modified at both ends
what happens to introns and exons in mRNA euk
introns removed via splicing, exons stay
where are euk mRNA transported from for translation
from nucleus to cytoplasm
RNA processing steps occur when
often occur concurrently with transcription
in bacterial mRNA processing, how are ends formed
5 and 3’ ends directly formed by transcription initiation and termination
no nucleus means what in bacterial mRNA processing
transcription and translation occur in the same compartment
in bacterial mRNA processing, what is special about timing of transcription and translation
translation can begin before transcription is done
how many proteins encoded in bacterial mRNA processing
multiple unlike eukaryotic mRNA
describe mRNA in bacteria
unmodified 5’ and 3’ ends, directly synthesized by RNA polymerase
describe eukaryotic mRNA
modified ends - 5’ cap added, 3’ end cleaved and poly A tail attached
key difference between euk and bacterial mRNA
bacterial mRNAs can encode multiple proteins while eukaryotic mRNAs typically encode only one
RNA splicing removes what from what
introns from pre mRNAs
Pre-mRNA Splicing Reaction
- An adenine nucleotide in the intron
attacks the 5′ splice site, cutting the RNA
backbone. - The cut 5′ end of the intron links to the
adenine, forming a loop (lariat structure). - The free 3′-OH end of the exon reacts with
the next exon, joining them together. - The intron is released as a lariat and later
degraded into single nucleotides for
recycling
what are the key sequences for intron removal with splicing
GU at the 5’ splice site, AG at the 3’ splice site, A forms branch point in the lariat structure during splcing
R represents what
purine (a or G)
Y represents what
pyrimidine (c or U
n represents what
several nucleotides that can occupy remaining positions
the branch point and 3’ splice junction are typically what than the 5’ spice site and branch pt
closer than
a single nucleotide change at splice site can cause what
mRNA with missing exon, mRNA with extended exon, mRNA with extra exon inserted between exons
True or False: During pre-mRNA splicing, the adenine nucleotide in the intron attacks the
3′ splice site, forming a loop and allowing the two exons to be joined together while the
intron is released as a lariat.
false
RNA splicing only happens in what
eukaryotic mRNA
Specific sequences in RNA direct what of the 3’ end
direct cleavage and polyadenylation
hexamer bound by CPSF
AAUAAA
what element on 3’ end beyond cleavage site is bound by CstF
GU-rich element
sequence recognized by another protein factor required for cleavage at 3’ end
CA sequence
DNA probes can bind to both RNA and DNA targets. How could you design a probe that
would bind to a gene’s DNA sequence but not its mRNA sequence? How could you design
a probe to bind to a gene’s mRNA sequence but not its DNA sequence?
1 - would need to target regions of the gene that are present in DNA but absent from mature mRNA like introns since they are removed during RNA splicing
2 - you need to target features unique to the mRNA - something like the poly A tail or a exon exon junction since it wouldn’t exist in the DNA
from RNA to protein main steps
initiation of protein synthesis, completion of protein synthesis and protein folding, protein degradation
mRNA sequence decoded in what
sets of 3 nucletides
codons always written with what on the left
5; terminal nucleotide on the left
most amino acids represented by what
multiple codons
codons from the same amino acid typically have the same what and different what
same first and second position, different last positon
how many codons serve at stop codons
3
2 functions of AUG
acts as initiation codon, codes for methionine
how many possible reading frames
3
translation reads mRNA in what direction
5’ to 3’
in theory the same RNA sequence can be translated in how many different frames
3
what is an anticodon
3 nucleotide sequence that bp with mRNA codon
amino acid is attached where in tRNA
3’ end
tRNAs do what
match amino acids to codons in mRNA
tRNAs contain what and give examples
unusual bases (pseudouridine, dihydrouridine
wobble bp occurs between what
codons and anticodons
what position is wobble position
third
wobble position allows for what
flexible bp
what does the flexible bp by wobble position enable
some tRNAs to recognize multiple codons for the same amino acid
in bacteria, wobble codon base is U, what are possible anticodon bases
A, G or I
in bacteria, wobble base is C, possible anticodon base
G or I
in bacteria, wobble base is A , what are possible anticodon base
U or I
In bacteria, wobble base is G, possible anticodon base
C or. U
in euk, wobble base is U, possible anticodon base
A, G, or I
in euk, wobble base is C, possible anticodon base
G or I
euk, wobble is A, anticodon base is
U
in euk, wobble eis G, anticodon base is
C
tRNA anticodon formed by deamination of adenosine
inosine
inosine can pair with what in bacteria and what in euk
bact - A, U, C euk - A or U
wobble base pairing is what compared to conventional bp
weaker
Aminoacyl-tRNA synthetases (aaRSs) do what
attach AAs to corresponding tRNAs
steps for aaRSs
Step 1: Activation – The amino acid’s carboxyl group binds to AMP, forming an adenylated amino acid.
This reaction is driven by ATP hydrolysis.
Step 2: Transfer to tRNA – The amino acid is transferred from AMP to the 3′ end of one the tRNAs that
can code for it. This forms a high-energy ester linkage, creating aminoacyl-tRNA.
: Incoming amino acid covalently linked to growing chain through a peptide bond,
catalyzed by the ribosome’s peptidyl transferase activity.
peptide bond formation
peptide bond formation catalyzed by what
ribosomes peptide transferase activity
High-energy peptidyl-tRNA bond drives …
peptide bond formation, making rxn thermodynamically favorable
With each addition, the peptidyl-tRNA linkage at the growing end is…
re-formed,
ensuring continuous elongation.
True/False: There is a different aminoacyl-tRNA synthetase for each anti-codon of the
genetic code
false
Holds the mRNA strand in place for
codon recognition.
mRNA binding site
3 tRNA binding sites ar what
A site, P site, E site
– The entry site for a
new tRNA carrying an amino acid.
Aminoacyl tRNA site
Holds the tRNA
linked to the growing polypeptide chain.
peptidyl tRNA site
Where the used tRNA, now
uncharged, is released from the ribosome.
exit site
2 subunits of ribosome
large and small ribosomal subunits
steps of translating an mRNA molecule
1.Aminoacyl-tRNA Binding –charged tRNA enters vacant A
site, matching its anticodon to the mRNA codon.
2.Peptide Bond Formation – The growing polypeptide
chain, attached to the tRNA in the P site, is transferred to
the amino acid on the tRNA in the A site via a peptide bond.
3.Large Subunit Translocation – The ribosome’s large
subunit moves forward, shifting tRNAs into new positions
4.Small Subunit Translocation – The small ribosomal
subunit shifts forward by three nucleotides, moving the
mRNA along with it. The uncharged tRNA is ejected from the
E site. The A site is ready. This “resets” the ribos
A complex formed by multiple
ribosomes translating the same mRNA molecule simultaneously
polyribosome
Ribosomes attach to the…
mRNA and move along it, synthesizing
polypeptides.
Multiple Ribosomes on One mRNA: explain
Several ribosomes bind to the
same mRNA, allowing for coordinated protein synthesis
efficiency of polyribosome
This parallel translation speeds up protein production
by allowing multiple copies of the protein to be synthesized from the
same mRNA
Spacing between Ribosomes
spaced at intervals
along the mRNA, ensuring efficient processing without
interference.
Different chaperones cooperate to ensure…
correct protein folding
Early-stage
chaperone that binds nascent polypeptides as they
emerge from the ribosome
Hsp70 (Heat Shock Protein 70 Family)
Recognizes exposed
hydrophobic regions, preventing improper folding or
aggregation.
hsp70
Functions after
Hsp70 to provide additional folding assistance.
Hsp90 (Heat Shock Protein 90 Family):
Chaperonins: Used when…
Hsp70 and Hsp90 are insufficient for proper folding
chaperonins provide what
secluded
environment where proteins can fold without interference from other cellular components.
Which of the following is the sequence of events in protein synthesis according to the translation cycle?
A) The small subunit translocates first, followed by the formation of a peptide bond and binding of the aminoacyltRNA.
B) The aminoacyl-tRNA binds to the E site, the peptide bond is formed, and the small subunit translocates.
C) The peptide bond is formed first, followed by the binding of the aminoacyl-tRNA and the translocation of the large
subunit.
D) The aminoacyl-tRNA binds to the A site, a peptide bond is formed, the large subunit translocates, and the small
subunit translocates to reset the ribosome.
E) None of the above.
D
why is gene expression control so inefficient in eukaryotes , but what purpose could this inefficiency serve
bc of variation of cell types (if it was efficient, all cells would be similar) but it is precise and flexible, allows specialization and development
why might euk be able to afford inefficiency in gene expression
euk cells have other cells that can pick up slack, for a single cell euk can afford it because of the mitochondria
what does euk gene expression control inefficientcy give rise to
regulation -> can be used in specialization/multicellularity
what do neuron and liver cells share but why are they different
same genome, express different RNAs and proteins
specialized functions come from what
selective gene activation
How do we know differentiated cells contain all the
information needed to make the entire organism?
adult frog skin cells put into an unfertilized egg with no nucleus, that cell developed into a normal embryo and a tadpole
in the frog experiment, what is the tadpole to the frog
a clone
Differences in RNA levels for two human genes in seven different tissues
*RNA collected from seven human cell lines was sequenced and mapped to the human genome.
*The height of the colored trace indicates the number of RNA sequences matching a genome region.
*Why is the number of reads different for the exons for the introns?
Exons have higher reads because they remain in mature mRNA, the reason for some reads in introns is from unspoiled premRNA
*The height of the colored trace indicates the number of
RNA sequences matching a genome region
what is actin
a cytoskeletal protein, is
expressed in all cells.
*How does the expression of Tyrosine
aminotransferase compare to the
expression of actin?
actin is expressed in a lot of cells because of it being a housekeeping gene, actin is expressed much less
7 steps at which euk gene expression can be controlled
transcriptional control , RNA processing control, RNA transport and localization control, translational control, mRNA degradation control, protein degradation control, protein activity control
when does RNA processing control occur
between RNA transcript and mRNA
when does RNA transport and localization control occur
when mRNA is transported from nucleus to cytosol
mRNA degradation control happens when
mRNA in cytosol and inactive mRNA
translational control occurs when
mRNA to protein
common promotor sequence is what
TATA box
where is TATa box
~30
nucleotides upstream of transcription start site
function of TATA box
Recruits the general transcription factors needed for
the initiation of transcription by RNA polymerase II
gene control regions regulate
transcription
DNA sequence where general transcription factors and RNA polymerase assemble
promotor
promotor is a DNA sequence where what
gen transcritpion factors and RNA polymerase assemble
how can gene control regions vary by location
can be near promotor, far upstream, within introns, downstream
Distance between cis-regulatory sequences and transcription start varies how
can be 10s of 1000s of nt pairs
sequence logo representation shows what
preferred nt for specific transcription factor binding
site for
transcriptional factor/regulator
cis regulatory sequence
Many transcription factors form…
homodimers or heterodimers
what makes up a dimer
2 transcription regulators together
True/False: cis-regulatory sequences can be located both upstream AND
downstream of the transcription start site and mediate control of gene
expression.
A) True
B) False
true
euk transcription regulators form what
multiprotein complexes
function of multi protein complexes
activate or repress transcription
describe coactivator and corepressor binding
do not directly bind DNA but assist transcription regulators
noncoding RNAs in some compelxes, describe function
scaffold to stabilize protein assemblies
The repressor can compete with the binding of
activator proteins for the same regulatory DNA sequence
The repressor can bind near the activator on DNA and…
interfere with activator function, such as blocking coactivator recruitment
The repressor can stabilize an…
intermediate in the transcription factor assembly process , preventing proper formation
True/False: The general transcription factors are a group of DNA binding
proteins that interact as homo- or hetero- dimers with cis-regulatory DNA
sequences outside the TATA box.
False
Each cell division leads to a decision to produce…
one of a pair of transcription regulators
Once initiated, each transcription regulator’s production
is…
self perpetrating
Cell memory allows…
combinatorial specification to develop step by step
How might you have different daughter cells from a single
cell division express different transcription factors?
asymmetric division, different signals received post division, epigenetic regulation
positive feedback loop enables what
cell memory
A feed-forward loop can measure
duration of a signal
Using Reporter Genes to Study DNA Regulatory Regions brief description
inserting a reporter gene next to a regulatory DNA sequence, its inserted into a cell or organism, if the regulatory region is active, then the expression of the reporter gene will be driven
what is a reporter gene
gene that produces an easily detectable product
which study of gene expression is better for studying regulatory elements
reporter gene method
Alteration of DNA Regulatory Regions in the reporter description and purpose
deliberately changing specific parts of a DNA reg sequence within a reporter context to study how those changes affect the reporter gene expression
common reporters
lacZ, GFP, luciferase
what is a recombinant in reporter gene study
fusion of upstream DNA region (reg seq) to a downstream reporter gene
reporter genes alone can determine what
sufficiency and requirement of upstream DNA reg regions (cis) in driving expression
reporter genes alone cannot determine what (why)
role of DNA reg regions (trans) outside of the tested region because reporters only provide insight into the reg region they are physically connected to in the test
what is sufficiency
region or factor is capable of initiating or driving gene exp on its own, without needing additional factors
what is requirement
reg region/factor is required if it is completely necessary for gene exp to occur
what is in situ hybridization
technique used to detect and localize specific mRNA molecules within tissue sections or whole organisms
what does in situ hybridization reveal
gene expression patterns by showing where and when specific genes are being transcribed in cells or tissues
alone, in situ hybridization can do what
determine place and timing of mRNA expression, also the overlap of mRNA exp with other mRNAS, cels, or tissues
in situ hybridization alone cannot determine..
protein expression, expression of 1000s of mRNAs simultaneously
brief description of bulk RNA seq
analyzes entire trasncriptome of a sample, showing gene expression over time
in bulk seq, what do columns mean
genes
in bulk seq, what do rows mean
samples/time
what is serum in bulk seq and what does it do
it is liquid part of blood after clotting, added to cell cultures or tissues in the experiment and can trigger gene expression, allows simulation of various physiological conditions
bulk RNA seq alone can do what
reveal genes differentially regulated across samples (time)
bulk seq alone cannot what
resolve hetergeneity in gene exp amongst cells within that sample
what can’t bulk rNA seq resolve heterogeneity
sample is composed of lots of different cells, results in an average
what is single cell RNA seq
measures gene exp at level of individual cells,
how does single cell RNA seq work
isolation of individual ells, RNA extracted, amplification and sequencing, analysis
in a single cell RNA seq plot, what does individual dots mean and what are the clouds
dots are individual cells,
clouds are groups of similar cells
single cell RNA seq alone can…
resolve all major mRNAs expressed in the individual cells from a whole animal, tissue, or organ
reveal heterogeneity of gene exp across individual cells
single cell RNA seq alone cannot..
distinguish boundary between cell types and states,
determine spatial information about cell types or exp patterns
why can’t single cell RNA seq determine spatial info about cell types or exp patterns
it captures gene exp data, not spatial context
You hypothesize that the mRNA of your gene of interest is expressed in a subset of cells in the
anterior region of the developing brain. Which technique would best test this hypothesis?
in situ hybridization
regulatory
mechanism for gene silencing and posttranscriptional control in eukaryotes.
RNAi system
Single-strand interfering RNAs (like siRNAs or miRNAs)
guide RNAi by
base pairing with complementary target RNAs
*Once bound, there are several possible outcomes: RNAi
- Cleavage and degradation of target mRNA
- Translational repression or target mRNA
- Heterochromatin formation on DNA from
which RNA is being transcribed
miRNAs are encoded where
genome
examples of single strand interfering RNAs
siRNAs or miRNAs
small regulatory RNAs used for post-transcriptional regulation of target mRNAs
microRNAs (miRNAs)
miRNAs are transcribed as what and form what
primary miRNAs, form hairpin structures
The Primary miRNA is processed into a… and where does this processing take place and where does it go
Precursor miRNA (Pre-miRNA) in nucleus and exported to cytosol
what cleaves it (pre-miRNA) to generate mature miRNA.
dicer enzyme
key protein in RISC
argonaute
where does argonauts bind
binds to both strands of the miRNA initially
what does argonauts do to miRNA
cleaves and discards one strand, uses the remaining strand to guide RISC to complementary mRNA targets
what does RISC stand for
RNA induced silencing complex
what determines the outcome of mRNA in other strands
extent of base pairing between miRNA and mRNA
*In plants: extensive base-pairing leads to
mRNA cleavage
and degradation
in plants, what is mRNA cleavage and degradation known as
slicing
*In mammals: partial base-pairing (typically a 7-nucleotide
“seed” region near the 5′ end of miRNA) results in:
- Inhibition of translation.
- Eventual mRNA degradation.
partial base pairing in mammals is typically what
7-nucleotide
“seed” region near the 5′ end of miRNA)
mature miRNA is how long
19-21 nt
single miRNA capable of affecting how many mRNA
1000s
miRNA act as what
tuners - modify gene expression
what enables selective genetic interference
Introduction of double-stranded RNA (dsRNA) or
synthetic small interfering RNAs (siRNAs) matching
specific targets
DsRNA can be made from vectors expressed in..
E. coli
and then fed to organisms like C. elegans
Question 1. A recombinant gene reporter in which the upstream DNA regulatory region of the gene
eve is fused to DNA containing the coding sequence for GFP can be regulated by miRNAs that
normally target eve. and why
A) True
B) False
false bc GGFP has different nucleotides than eve
enabled the study of gene function in a wide
variety of species.
CRISPR
step 1 in CRISPR mediated immunity
short viral DNA sequence is integrated into CRISPR locus
step 2 in CRISPR mediated immunity
RNA transcribed form CRISPR locus, processed, bound to Cas protein
step 3 of CRISPR mediated immunity
small crRNA in complex with Cas seeks out and destroys viral sequences
CRISPR mediated immunity: A small DNA fragment from the virus is
inserted into the host’s…
CRISPR locus
what fraction of infected cells survive viruses initial attack
small fraction
transcript of CRISPR locus processed into what
crRNAs
what does crRNA stand for
CRISPR RNAs
. If the population is reinfected by the same
virus,.. what happens
crRNAs recognize the viral DNA, complementary viral DNA is destroyed
Cas9 used for what
defending against viruses
Cas9 protein is artificially expressed in the…
target organism
Cas9 binds a
guide RNA
what is guide RNA composed of
region required for Cas9 binding and sequence matching specific genome target
The gRNA targeting sequence is designed by
the experimenter
Cas9 creates what at the targeted genomic site
double stranded break
double strand break created by Cas9 usually repaired by what, can introduce what
repaired by non homologous end joining, can introduce mutations
what can be used for precise gene editing in Cas 9 protein break , what is needed to use it
homologous recombination, needs an altered repair template
mutant Cas9 would not have what ability
cannot cleave DNA
mutant Cas9 can be used for what
activation and repression
mutant Cas9 fused to activator can do what
turn on dormant genes
mutant cas9 fused to a repressor does what
silence active genes
Question 2. RNAi can be used to generate specific gene deletions and facilitate homologous
recombination. why
A) True
B) False
false, it silences gene expression by degrading mRNA or blocking its translation
what can be introduced into cultured embryonic stem cells in generation of transgenic mice from modified embryonic stem cells
an altered genes
what replaces the normal gene with the altered gene in modified stem cells
homologous recombination
how are modified embryonic stem cells identified
using selection marker and PCR
what happens to cells who show the selection marker in PCR for modified embryonic stem cells
cultured to produce ES cell lines carrying the altered gene
Altered ES cells are injected into
an early mouse embryo
ES cells once injected into embryo do what
creating a chimera with some altered somatic cells.
some mice will have what kind of cells that carry the altered gene, which is ideal
germ line cells
If the germline has been modified, when bred with normal
mice, what happens
some offspring inherits one copy of the altered gene in all cells
These heterozygous mice (one normal, one mutant gene)
are bred to produce
both male and female cariiers
When two heterozygous mice are mated, what percent of their offspring will be homozygous for altered gene
1/4
Question 3. Once injected into the early embryo, genetically modified stem cells can incorporate
into either somatic or germline tissues, with germline incorporation being the ideal outcome.
A) True
B) False
true
