1
Q
A
2
Q
What is the difference between qualitative and quantitative molecular analysis?
A
Qualitative identifies size, structure, or sequence; quantitative measures how much of a gene product is present.
3
Q
What is a molecular probe?
A
A labeled DNA or RNA sequence that binds a complementary target to detect it.
4
Q
How are oligonucleotide probes labeled?
A
Polynucleotide kinase (PNK) adds a radioactive or fluorescent phosphate to the 5′ end.
5
Q
When do you use a Southern blot?
A
To detect specific DNA sequences. Example: checking if a mutation removes an EcoRI restriction site.
6
Q
When do you use a Northern blot?
A
To detect RNA (mRNA) levels and transcript sizes. Example: measuring expression of a gene to see if it is turned on in a tissue.
7
Q
What does Southern blotting detect?
A
DNA sequence changes, insertions/deletions, or polymorphisms.
8
Q
What does Northern blotting detect?
A
mRNA abundance and transcript size differences.
9
Q
What does qPCR measure?
A
The amount of cDNA produced during amplification, reflecting original mRNA levels.
10
Q
What converts mRNA into cDNA before qPCR?
A
A reverse transcriptase reaction (RT).
11
Q
What does a lower Ct value mean in qPCR?
A
More starting cDNA/mRNA (higher gene expression).
12
Q
What percentage of gene expression regulation occurs at transcription?
A
About 73%.
13
Q
What strand direction does RNA polymerase read during transcription?
A
3′ to 5′ on the template strand.
14
Q
In what direction is RNA synthesized?
A
5′ to 3′.
15
Q
What is the +1 site?
A
The transcription start site.
16
Q
What are promoter sequences?
A
Upstream regulatory DNA that recruits RNA Polymerase II.
17
Q
What are the three stages of transcription?
A
Initiation, elongation, termination.
18
Q
What type of mRNA do prokaryotes produce?
A
Polycistronic mRNA (multiple proteins per transcript).
19
Q
What type of mRNA do eukaryotes produce?
A
Monocistronic mRNA (one protein per transcript).
20
Q
What does RNA Polymerase I synthesize?
A
rRNA in the nucleolus.
21
Q
What does RNA Polymerase II synthesize?
A
mRNA, miRNA, siRNA.
22
Q
What does RNA Polymerase III synthesize?
A
tRNA, 5S rRNA, U6 snRNA, 7S RNA.
23
Q
What toxin inhibits RNA Polymerase II?
A
Amanitin.
24
Q
What is the CTD of RNA Pol II?
A
A tail of heptapeptide repeats on the large subunit, heavily phosphorylated during transcription.
25
What is the function of CTD phosphorylation?
Recruits capping, splicing, and polyadenylation factors during active transcription.
26
What are chromosome puffs?
Regions of decondensed chromatin representing highly active transcription.
27
What causes chromosome puffs to form?
Accumulation of RNA Pol II with phosphorylated CTD and high transcriptional activity.
28
What is an example of puff formation?
Heat-shock puffs in Drosophila where HSP genes are rapidly transcribed.
29
What is the major level of gene expression control?
Transcription (about 73% of regulation).
30
What is a key similarity between prokaryotic and eukaryotic transcription?
Both need promoters, multi-subunit RNA polymerases, and DNA-binding regulatory proteins.
31
What is the major difference in transcription between prokaryotes and eukaryotes?
Prokaryotes couple transcription and translation; eukaryotes separate them due to the nucleus.
32
What gives promoter specificity in prokaryotes?
Sigma (σ) factors.
33
Why is chromatin important in eukaryotic transcription?
Its condensation or decondensation regulates RNA polymerase access.
34
What is the TATA box?
A promoter element that positions Pol II; present in some but not all genes.
35
What do activators do?
Bind promoters/enhancers and decondense chromatin to stimulate transcription.
36
What do repressors do?
Condense chromatin or block transcription factor access.
37
What is special about Pax6 regulation?
It uses multiple promoters and many enhancers for tissue-specific expression (eye, CNS, pancreas).
38
What does comparative genomics reveal about enhancers?
Highly conserved enhancers can lie tens or hundreds of kb away from the gene.
39
What does RNA Pol I synthesize?
Major rRNAs (28S, 18S, 5.8S).
40
What does RNA Pol II synthesize?
mRNA, miRNA, siRNA, snRNA, lncRNA.
41
What does RNA Pol III synthesize?
tRNA, 5S rRNA, U6 snRNA, 7S SRP RNA.
42
Which polymerase is inhibited by α-amanitin?
RNA Polymerase II (highly sensitive).
43
What is the clamp domain of Pol II responsible for?
Stabilizing DNA and RNA during elongation for high processivity.
44
What is the CTD of Pol II?
A tail of heptad repeats (YSPTSPS) on RPB1 that becomes phosphorylated during active transcription.
45
What does CTD phosphorylation do?
Recruits capping, splicing, and polyadenylation factors during elongation.
46
What do polytene chromosome puffs represent?
Regions of highly active transcription enriched for phosphorylated Pol II CTD.
47
How are recombinant DNA techniques used in regulation studies?
DNA fragments are cloned and introduced into cells/organisms to test function.
48
What is a reporter assay used for?
To measure the activity of promoters or enhancers by linking them to a measurable reporter gene.
49
How does a transient transfection assay work?
A regulatory DNA fragment is cloned upstream of a reporter, transfected into cells, and reporter output indicates regulatory activity.
50
What provides promoter specificity in prokaryotic transcription?
Sigma (σ) factors.
51
What provides promoter specificity in eukaryotic transcription?
General transcription factors (GTFs), especially TFIID/TBP.
52
What is the first general transcription factor to bind a Pol II promoter?
TFIID (TBP + TAFs).
53
What does TBP do to DNA when it binds the TATA box?
Binds the minor groove and sharply bends the DNA.
54
What is the correct order of PIC assembly at a TATA box promoter?
TFIID → TFIIA → TFIIB → Pol II/TFIIF → TFIIE → TFIIH.
55
What factor melts DNA during promoter opening?
TFIIH helicase XPB, using ATP.
56
What does TFIIH kinase do during initiation?
Phosphorylates Ser5 of the Pol II CTD, recruiting capping enzymes.
57
What is the function of CTD Ser2 phosphorylation?
Promotes elongation by recruiting splicing and polyadenylation factors.
58
What evidence shows CTD phosphorylation marks active transcription?
Polytene chromosome puffs stain strongly for phosphorylated CTD.
59
What is the purpose of ChIP?
To identify genome-wide binding sites of proteins such as Pol II or TBP.
60
What are the core steps of a ChIP experiment?
Crosslink → sonicate → immunoprecipitate → reverse crosslinks → analyze DNA.
61
What causes promoter-proximal pausing of Pol II?
DSIF + NELF binding to Pol II, blocking productive elongation.
62
What releases Pol II from pausing?
P-TEFb (CDK9–cyclin T) phosphorylates NELF, DSIF, and CTD Ser2.
63
What prevents NELF from re-binding after pause release?
SPT6 and the PAF complex.
64
What does DSIF become after phosphorylation by P-TEFb?
A positive elongation factor.
65
How does HIV Tat stimulate transcription?
Tat binds TAR RNA and recruits P-TEFb, enhancing elongation.
66
What is a CpG island promoter?
A broad, TATA-less promoter that supports weak, divergent transcription.
67
What is the role of recombinant DNA in regulatory studies?
Allows cloning and manipulation of regulatory sequences to test their function.
68
What is a transient transfection assay used for?
To test promoter or enhancer activity by driving a reporter gene.
69
How do enhancers differ in location compared to promoters?
Enhancers can act tens or hundreds of kilobases from the gene.
70
What are the main cis-acting regulatory elements in eukaryotic genes?
Promoters, promoter-proximal elements, and enhancers.
71
How can subregions of a promoter required for transcriptional activation be identified?
Using linker scanning mutations.
72
Name common reporter genes used to quantify transcriptional activity.
GFP, β-galactosidase (lacZ), thymidine kinase (tk), luciferase (luc), chloramphenicol acetyltransferase (CAT).
73
What is the purpose of an electrophoretic mobility shift assay (EMSA)?
To detect DNA binding by transcription factors; protein-DNA complexes migrate more slowly in non-denaturing gels.
74
Can EMSA determine the exact DNA sequence a transcription factor binds?
No, it shows binding activity but not the precise sequence.
75
How do transcription factors recognize DNA?
Via an α-helical recognition helix making non-covalent contacts with base atoms in the major groove.
76
What is an example of a transcription factor that binds DNA as a homodimer?
Bacteriophage 434 repressor.
77
What does it mean that transcription factors are modular?
They have separate domains for DNA-binding, activation, repression, chromatin remodeling, nuclear import, and protein-protein interactions.
78
Give an example of a modular transcription factor and its domains.
GAL4 in yeast: DNA-binding domain binds UAS^GAL; activation domain stimulates transcription.
79
What is the homeodomain?
A DNA-binding domain in transcription factors regulating developmental genes.
80
What can mutations in homeodomain proteins cause?
Homeotic transformations (changing identity of body segments).
81
How do combinatorial interactions affect transcription?
Transcription factors can repress or activate genes depending on interactions, creating complex regulatory patterns.
82
Give an example of cooperative transcription factor binding.
NFAT and AP1 cooperate at a promoter-proximal element of the IL-2 gene; individually unstable, together they form a ternary complex.
83
What is cooperative binding in transcription regulation?
Some factors bind DNA only when neighboring sites are occupied by interacting factors.
84
Give an example of cooperative binding with distant DNA sites.
SRF and SAP1 bind cooperatively even if sites are 5–30 bp apart or inverted, using flexible linkers.
85
What are enhanceosomes?
Enhancers (~50–200 bp) where multiple transcription factors assemble into a DNA-protein complex to regulate transcription.
86
Give an example of an enhanceosome.
β-interferon enhancer binds Jun/ATF-2, p50/RelA (NF-κB), IRF-3, and IRF-7 to form a large regulatory complex.
87
How do flexible linkers and DNA looping affect transcription regulation?
They allow distant transcription factors to interact, increasing regulatory flexibility and evolutionary diversity.
88
What is the role of the Mediator complex in transcription?
Mediator bridges enhancer-bound transcription factors and the basal transcription machinery (RNA Pol II), facilitating transcription initiation over long chromatin distances.
89
What are the main structural domains of the Mediator complex?
Head (binds DNA-binding activators), middle, and tail (binds DNA-binding activators).
90
Are Mediator subunits essential?
Yes, mutations in many Mediator subunits are lethal.
91
What characterizes highly transcribed genes?
High transcriptional activation, rapid initiation, high burst frequency of RNA Pol II, not just RNA abundance.
92
What is a 'reporter gene' strategy for measuring transcriptional efficiency?
Place a reporter downstream of a promoter; RNA structure is recognized by a fused RNA-binding protein (e.g., GFP), allowing direct measurement of transcription in real time.
93
What is the significance of the 5' end RNA folding in reporters?
The 5' RNA forms hairpin structures recognized by RNA-binding proteins, enabling visualization of newly transcribed RNA.
94
What is the transcriptional bursting phenomenon?
Transcription occurs in successive bursts rather than a constant flux; frequency of bursts correlates with overall transcription efficiency.
95
What is a shadow enhancer?
A secondary enhancer ensuring correct temporal and spatial transcription, often increasing burst frequency without changing amplitude.
96
What are P-granules and their significance?
Liquid droplets in embryos rich in RNA; specify germ cell lineage; posterior P-granules condense, anterior remain soluble.
97
How do modular transcription factor domains contribute to transcription?
Domains allow DNA binding, activation, repression, chromatin remodeling, nuclear import, and protein-protein interactions.
98
What are intrinsically disordered regions (IDRs) and their role in transcription?
Protein regions that can form liquid-liquid condensates, helping concentrate transcription machinery and regulators.
99
What factors promote liquid-liquid condensate formation in transcription?
Macromolecule concentration (DNA, RNA, protein), valency, electrostatic interactions, post-translational modifications, and intrinsically disordered protein regions.
100
How do Mediator and RNA Pol II behave in the nucleus?
They form liquid-like condensates, dynamically assembling and disassembling during transcription initiation ('dynamic kiss' model).
101
What is the RNA-mediated feedback control model of transcriptional condensates?
RNA produced at enhancers and promoters acts as a glue to maintain condensates; when RNA reaches a threshold, condensate dissolves, releasing Pol II in a burst.
102
How does enhancer positioning affect transcriptional bursts?
Position affects efficiency; 5' enhancers may produce stronger bursts than 3' enhancers, but both contribute to proper regulation.
103
What determines transcriptional burst frequency and amplitude?
Burst frequency is modulated by enhancer strength and availability of transcription factors; amplitude is influenced by Pol II activity within each burst.
104
How does chromatin act as a barrier to transcription?
Chromatin restricts DNA access by wrapping DNA around nucleosomes; transcription requires histone modifications or remodeling to allow factor access.
105
What effect does histone acetylation have on chromatin structure?
Acetylation neutralizes positive charges on histone tails, loosening histone-DNA interactions and allowing transcription factor binding.
106
What role do histone deacetylases (HDACs) play in transcriptional repression?
HDACs remove acetyl groups from histone tails, maintaining compact chromatin and repressing transcription.
107
Name examples of co-repressors and their mechanisms.
Rpd3p recruited by Ume6p and Sin3p in yeast; Sir2 deacetylates histones to maintain repression.
108
How do transcriptional activators promote gene expression?
Activators recruit HATs and chromatin remodelers (SWI/SNF) to open chromatin and facilitate transcription factor access.
109
Give examples of co-activators in yeast and mammals.
Yeast: Gcn4p recruits Gcn5p; Mammals: CBP, p300.
110
What are pioneer transcription factors?
DNA-binding factors that bind nucleosomal DNA and recruit HATs to open chromatin, allowing general transcription factors access.
111
Why are pioneer factors important?
Essential for early embryogenesis and activation of gene regulatory networks.
112
What is the histone code?
Specific histone tail modifications correlate with chromatin states, e.g., H3K4me = active, H3K9me = repressed.
113
Can histone modifications always be generalized?
No, context matters; the same modification may have different effects depending on location and interacting factors.
114
What is chromatin immunoprecipitation (ChIP)?
Technique using antibodies against histones or transcription factors to identify DNA regions bound by these proteins.
115
What are key steps in ChIP?
1. Crosslink protein-DNA, 2. Immunoprecipitate with antibodies, 3. Identify DNA via PCR or NGS.
116
What are epigenetic marks?
Heritable traits independent of DNA sequence, such as histone modifications or DNA methylation.
117
Give examples of epigenetic processes.
Inactive X chromosome (Xist, heterochromatin), developmental fate decisions (Polycomb proteins), genomic imprinting (DNA methylation).
118
How are epigenetic marks propagated?
Histone marks recruit enzymes that write the same mark on neighboring histones; DNA methylation recruits chromatin-modifying proteins.
119
Differentiate epigenetic readers and writers.
Writers establish marks (e.g., HMTs); Readers recognize and interpret marks; some proteins function as both.
120
What is the role of H3K9me3 in heterochromatin?
Critical for propagation during DNA replication; recognized by HMTs that methylate adjacent naïve histones.
121
Why are chromatin-modifying enzymes essential for transcription regulation?
They enable activators and repressors to alter chromatin accessibility, controlling gene expression.
122
Summarize the key functions of co-repressors vs co-activators.
Co-repressors (HDACs) → compact chromatin, repress transcription; Co-activators (HATs, remodelers) → decondense chromatin, activate transcription.
123
What are the four main takeaways regarding chromatin and transcription regulation?
1. Chromatin is a transcription barrier, 2. TFs work with chromatin modifiers, 3. Pioneer factors open chromatin early, 4. Epigenetic marks guide cell fate and are heritable.
124
What is the spliceosome?
A dynamic ribonucleoprotein complex that removes introns and ligates exons via two transesterification reactions.
125
Which snRNAs form snRNPs in the spliceosome?
U1, U2, U4, U5, U6
126
What happens in the E complex of splicing?
U1 binds the 5′ splice site; SF1 and U2AF recognize the branch point and 3′ splice site.
127
What happens in the A complex of splicing?
U2 binds the branch point A with ATP-dependent help from Sub2 and Prp5.
128
What is the pre-B/B complex?
U4/U6.U5 tri-snRNP joins; Prp28 and ATP displace U1 to form the B complex.
129
What occurs in the activated B (Bact) complex?
U4 dissociates; Brr2 unwinds U4/U6; active catalytic center forms with U2 and U6.
130
What is the branching reaction (step 1)?
2′-OH of branch point A attacks the 5′ splice site forming a lariat intron.
131
What is step 2 of splicing?
Exon ligation: 5′ and 3′ exons are joined; lariat intron remains.
132
What happens during ILS disassembly?
Prp43 with ATP dissociates NTC/NTR and recycles snRNPs.
133
What is the function of the Exon Junction Complex (EJC)?
Forms ~20 nt upstream of exon junctions; assists nuclear export and NMD.
134
What is trans-splicing?
Splicing of exons from separate RNA molecules (e.g., *C. elegans*).
135
What signals define polyadenylation sites?
AAUAAA upstream; GU- or U-rich downstream element.
136
What proteins bind the poly(A) signal?
CPSF binds AAUAAA; CstF binds downstream GU/U element; CFI/CFII stabilize complex.
137
What is the role of PAP in polyadenylation?
Cleaves RNA at poly(A) site and adds initial A residues.
138
What is the role of PABPN1?
Binds first ~12 A residues and stimulates processive polyadenylation (~200–250 A).
139
What is Alternative Polyadenylation (APA)?
Use of multiple poly(A) sites in a gene; can change 3′UTR length and generate protein isoforms.
140
How is transcription coupled to RNA processing?
Pol II CTD Ser5-P recruits capping enzymes; Ser2-P recruits splicing and cleavage/polyadenylation factors.
141
What signals transcription termination?
Cleavage at the poly(A) site; downstream RNA is degraded by XRN1.
142
What is the role of the Sex-lethal (Sxl) gene in Drosophila?
Sxl is expressed only in females early in embryogenesis and regulates female-specific alternative splicing of downstream genes, ensuring sex-specific development.
143
What controls early Sxl expression?
An early promoter active only in females during embryogenesis.
144
What happens at the late Sxl promoter?
It is active in both sexes, but functional Sxl protein is only produced in females due to female-specific splicing.
145
Which downstream genes are regulated by Sxl?
TRA and TRA2, leading to sexually dimorphic characteristics.
146
How does Doublesex transcription factor contribute to sex determination?
Alternative splicing regulated by Sxl and TRA/TRA2 produces male or female isoforms.
147
What is an extreme example of alternative splicing?
Cochlear Ca²⁺-activated K⁺ channel pre-mRNA can be spliced in 576 ways, producing position-specific isoforms along the cochlea.
148
What is RNA editing?
Alteration of pre-mRNA sequence so that mature mRNA differs from the genomic DNA.
149
What are the common types of RNA editing?
Cytosine → Uracil (C → U) and Adenosine → Inosine (A → I).
150
Where is RNA editing most frequent?
Mitochondria and plastids of protozoans and plants; rare in nuclear genomes of higher eukaryotes.
151
Give an example of RNA editing in mammals.
ApoB pre-mRNA: liver retains CAA codon → apoB-100; intestine edits CAA → UAA stop codon → apoB-48.
152
What is the functional consequence of RNA editing?
Single-base changes can dramatically alter protein length and function.
153
What is the purpose of mRNA polyadenylation?
Stabilizes the 3′ end of mRNAs and prevents rapid degradation.
154
Which mRNAs are exceptions to polyadenylation?
Histone mRNAs lack poly(A) tails but are stabilized by 3′ UTR secondary structures.
155
Describe the mechanism of polyadenylation.
Pol II transcribes past AAUAAA signal → cleavage downstream → slow addition of ~12 A by PAP → PABPN1 stimulates rapid addition to ~200 A → CPSF coordinates cleavage/polyadenylation.
156
Key proteins involved in polyadenylation?
PAP, PABPN1, CPSF.
157
Why is alternative splicing important?
It allows multiple mRNAs to be produced from a single gene.
158
What does Sex-lethal regulation exemplify?
Post-transcriptional control producing sex-specific phenotypes.
159
Why is RNA editing functionally important despite being rare?
It produces nucleotide-specific RNA modifications that can alter protein function.
160
Why is polyadenylation critical?
It ensures mRNA stability and proper translation; histone mRNAs are an exception.
161
What separates the nucleus from the cytoplasm?
The double-membrane nuclear envelope.
162
What mediates transport between nucleus and cytoplasm?
Nuclear pore complexes (NPCs).
163
What state do nuclear proteins enter the nucleus in?
Folded state.
164
What is the size limit for passive diffusion through NPCs?
≤40 kDa.
165
What is required for active nuclear transport?
Nuclear transport receptors (importins/exportins) and energy via Ran GTPase.
166
What sequence directs proteins to the nucleus?
Nuclear localization signal (NLS).
167
Which cytosolic factors are essential for nuclear import?
Importin and Ran GTPase.
168
What is the function of Ran GTPase?
Provides directionality and energy for nuclear import/export via its GTP/GDP cycle.
169
What are the key steps of importin-mediated nuclear import?
1. Importin binds NLS cargo; 2. Passes through NPC via FG-repeats; 3. Ran·GTP binds importin → cargo released; 4. Importin-Ran·GTP recycled to cytoplasm.
170
What sequence directs proteins for nuclear export?
Nuclear export signal (NES).
171
What is the mechanism of Ran-dependent nuclear export?
Exportin + Ran·GTP + NES cargo form a complex → diffuses through NPC → Ran-GAP hydrolyzes GTP → cargo released → exportin/Ran·GDP recycled.
172
How are mRNAs exported from the nucleus?
Ran-independent via NXF1/NXT1 heterodimer; released by Dbp5 helicase at cytoplasmic filaments.
173
What ensures only fully spliced mRNAs are exported?
RNA helicase-mediated remodeling.
174
What are Balbiani rings used for?
Visualization of transcription and mRNP export in polytene chromosomes.
175
Compare importin-mediated import vs exportin-mediated export.
Import: Ran·GTP triggers release; high cytoplasmic cargo; NLS proteins. Export: Ran·GTP part of complex; high nuclear cargo; NES proteins.
176
What is the purpose of the IRE–IRE-BP system?
Maintain intracellular iron at safe levels by regulating translation and mRNA stability
177
What proteins act as iron sensors in cells?
IRE-BPs (IRE-BP1/aconitase 1 from ACO1 and IRE-BP2 from IREB2)
178
What structural feature allows IRE-BPs to sense iron?
4Fe–4S iron-sulfur cluster
179
What happens to IRE-BPs when iron is low?
Fe–S cluster dissociates and IRE-BPs bind IREs in mRNAs
180
What is an IRE?
A stem-loop RNA structure in the UTR of iron-regulated mRNAs
181
Where are IREs located in ferritin mRNA?
5′ untranslated region
182
Effect of low iron on ferritin translation?
IRE-BP binds 5′ IRE and blocks ribosomal scanning → translation inhibited
183
Why inhibit ferritin at low iron?
To prevent iron storage and keep iron available for enzymes
184
Effect of high iron on ferritin translation?
IRE-BPs do not bind → ferritin translated → iron stored safely
185
What is ferritin?
A 24-subunit protein complex that stores excess iron
186
Where are IREs located in transferrin receptor (TfR) mRNA?
3′ untranslated region
187
Effect of low iron on TfR mRNA?
IRE-BP binding stabilizes mRNA → increased iron uptake
188
Effect of high iron on TfR mRNA?
IRE-BPs dissociate → AU-rich elements trigger mRNA degradation
189
Key contrast ferritin vs TfR regulation?
Ferritin controlled at translation; TfR controlled at mRNA stability
190
What are ribosomes?
Large ribonucleoprotein complexes that synthesize proteins
191
Ribosome subunits?
Small subunit decodes mRNA; large subunit catalyzes peptide bonds
192
Which molecules form the catalytic core of the ribosome?
rRNAs (ribosome is a ribozyme)
193
Accessory rRNAs in eukaryotic large subunit?
5S and 5.8S rRNA
194
Role of ribosomal proteins?
Mostly peripheral; stabilize rRNA structure
195
First step of eukaryotic initiation?
Assembly of 40S with eIF1
196
What is the 43S preinitiation complex?
40S + eIF2-GTP + Met-tRNAi + eIF5
197
Which tRNA initiates translation?
Initiator methionine tRNA (Met-tRNAi)
198
How is mRNA recruited to the ribosome?
eIF4 complex binds 5′ cap and poly(A) tail
199
What is scanning?
40S ribosome moves 5′→3′ to find AUG start codon
200
What triggers start codon recognition?
GTP hydrolysis by eIF2
201
What complex forms after AUG recognition?
48S initiation complex
202
How is the 80S ribosome formed?
60S joins after eIF5B-GTP hydrolysis
203
Key irreversible step in initiation?
Joining of 60S subunit
204
What factor delivers aa-tRNA to the A site?
EF1α-GTP
205
What ensures correct codon-anticodon pairing?
GTP hydrolysis by EF1α
206
What catalyzes peptide bond formation?
Large subunit rRNA
207
Which factor drives ribosome translocation?
EF2-GTP
208
What happens during translocation?
Ribosome moves one codon; A→P
209
From which site does tRNA exit?
E site
210
Where does the nascent polypeptide exit?
Exit tunnel in large subunit
211
What recognizes stop codons?
eRF1
212
What promotes peptide release?
eRF3-GTP
213
What releases ribosomal subunits after termination?
ABCE1 ATPase
214
What factors help recycle the 40S subunit?
eIF1
215
What is a polysome?
Multiple ribosomes translating one mRNA
216
Why does mRNA circularization matter?
Enhances ribosome recycling and translation efficiency
217
Which proteins mediate mRNA looping?
eIF4E/eIF4G and PABPC
218
What is a nonsense mutation?
Premature stop codon
219
What is a suppressor tRNA?
tRNA that reads through a stop codon
220
Effect of suppressor tRNAs?
Partial restoration of full-length protein
221
Role of small RNAs at centromeres?
Establish heterochromatin and silence transcription
222
Key histone mark in centromeric heterochromatin?
H3K9me3
223
What organism demonstrated RNA-based centromere silencing?
Schizosaccharomyces pombe
224
What is XIST?
A long non-coding RNA
225
Function of XIST?
Silences one X chromosome in cis
226
How does XIST act?
Spreads along X and recruits repressive chromatin modifiers
227
Result of XIST activity?
Stable epigenetic gene silencing
228
Core catalyst of translation?
rRNA
229
Energy source for translation checkpoints?
GTP hydrolysis
230
Purpose of non-coding RNAs in chromatin?
Gene silencing and epigenetic regulation
231
What is CRISPR-Cas9?
A programmable system that uses sgRNA-guided Cas9 to cut specific DNA sequences.
232
What usually happens after Cas9 cuts DNA?
Small insertions or deletions that disrupt gene function.
233
What enables precise genome editing with CRISPR?
Providing a donor DNA template for repair.
234
What is the purpose of genome-wide CRISPR screens?
Identify genes involved in a phenotype in one pooled experiment.
235
What does sgRNA enrichment after treatment indicate?
Gene knockout provides resistance or advantage.
236
What is dCas9?
Catalytically inactive Cas9 used for gene regulation.
237
What is CRISPRi?
dCas9-mediated transcriptional repression.
238
What is CRISPRa?
dCas9 fused to activators to increase transcription.
239
What is the loxP-Cre system used for?
Tissue-specific gene knockout in mice.
240
What is RNA interference (RNAi)?
Gene silencing by targeted mRNA degradation.
241
What enzyme initiates RNAi?
Dicer.
242
What complex degrades target mRNA in RNAi?
RISC.
243
What is the modular nature of transcription factors?
Separate DNA-binding and activation domains.
244
What does proteomics study?
Proteins, their abundance, modifications, and interactions.
245
What technique identifies proteins in proteomics?
LC-MS/MS.
246
What is proteomics?
The global study of protein abundance, modifications, interactions, and localization.
247
What key problem does proteomics solve?
It provides a global view of proteins rather than one at a time.
248
What core technique underlies modern proteomics?
LC-MS/MS (liquid chromatography–tandem mass spectrometry).
249
What is the basic workflow of LC-MS/MS?
Proteins are digested into peptides, separated by LC, and identified by MS/MS.
250
What does proximity-dependent labeling measure?
Proteins located near a target protein inside living cells.
251
Name one proximity-labeling method.
BioID, APEX, or TurboID.
252
What is phosphoproteomics?
The study of phosphorylated proteins involved in signaling.
253
Why is phosphoproteomics important?
Phosphorylation regulates signaling and protein activity.
254
What is CRISPR?
A bacterial adaptive immune system adapted for genome editing.
255
What directs Cas9 to a specific DNA sequence?
A single guide RNA (sgRNA).
256
What sequence is required for Cas9 binding?
A PAM sequence (NGG).
257
What type of DNA break does Cas9 create?
A double-strand break.
258
What are two major applications of CRISPR?
Gene knockout and precise genome editing.
Biol 112
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