Exam 3 Flashcards
(95 cards)
1
Q
- Which of the following is not true concerning eukaryotic mRNA processing?
A. addition of a 3 poly-A tail B. addition of a 5 cap C. intron splicing D. occurs in the nucleus E. occurs in the cytoplasm
A
E. occurs in the cytoplasm
2
Q
- The first two bases and the last two bases in the splicing signal consensus sequence are
A. GT-AG. B. GU-AG. C. CU-AG. D. GU-AC. E. GT-TG.
A
B. GU-AG.
3
Q
- Which of the following is the name for the Yeast 40S particle where mRNA splicing takes place?
A. ribosome B. nucleolus C. spliceosome D. hnRNA E. R-loop
A
C. spliceosome
4
Q
- “Snurps” are composed of
A. RNA. B. protein. C. DNA. D. RNA and protein. E. DNA and RNA.
A
D. RNA and protein.
5
Q
- Which of the following snRNP is mismatched with its function?
A. U1: base pairs with 5 splice site of mRNA.
B. U2: base pairs with the conserved sequence at splicing branch point.
C. U4: base pairs with 3 splice site of mRNA.
D. U5: associates with last nucleotide in one exon and the first nucleotide in the next exon.
E. U6: base pairs with 5 end of the intron.
A
C. U4: base pairs with 3 splice site of mRNA.
6
Q
- Which of the following splicing factors is required for the correct 3 splice site selection?
A. SC35 B. Slu7 C. U11 D. SF2 E. SRp55
A
B. Slu7
7
Q
- The catalytic center of the spliceosome appears to include
A. Mg2+.
B. U2 and U6 snRNP.
C. the branch point region of the intron.
D. Mg2+ and the branch point region of the intron.
E. Mg2+, U2, and U6 snRNP, and the branch point region of the intron.
A
E. Mg2+, U2, and U6 snRNP, and the branch point region of the intron.
8
Q
- Which of the following is the first snRNP to bind during the assembly stage of the spliceosome cycle?
A. U1 B. U2 C. U4 D. U5 E. U6
A
A. U1
9
Q
- The binding of which of the following snRNPs to spliceosome requires ATP?
A. U1 B. U2 C. U4 D. U5 E. U6
A
B. U2
10
Q
- The expressed regions of genes are referred to as ____ while the noncoding regions are called ____.
A
exons; introns
11
Q
- The process by which introns are removed from immature mRNA is called ____.
A
RNA splicing
12
Q
- Some introns, such as Group II introns, are self splicing. These types of introns contain catalytic RNA’s, also referred to as ____.
A
ribozymes
13
Q
- T/F: RNA polymerase II does not transcribe through introns, instead it jumps over them.
A
False
14
Q
- T/F: Heterogeneous nuclear RNA (hnRNA) is thought to be a precursor of mRNA.
A
True
15
Q
- T/F: The spliceosome cycle is one way for a cell to regulate gene expression.
A
True
16
Q
- T/F: Alternative splicing patterns can lead to the production of different protein products from the same gene.
A
True
17
Q
- Please put the following steps of Cap synthesis in the correct order.
(1) N7 of the capping guanine is methylated.
(2) The terminal phosphate is removed from the pre-mRNA.
(3) A capping GMP is added to the pre-mRNA.
(4) The 2-O-methyl group of the penultimate nucleotide is methylated.
A. 1, 2, 3, 4 B. 1, 4, 3, 2 C. 2, 4, 1, 3 D. 2, 3, 1, 4 E. 4, 3, 2, 1
A
D. 2, 3, 1, 4
18
Q
- Which of the following is NOT a function of the mRNA cap?
A. protects the mRNA from degradation
B. enhances translatability of the mRNA
C. enhances transport of the mRNA to the cytoplasm
D. enhances splicing of the mRNA
E. helps regulate expression of the mRNA
A
E. helps regulate expression of the mRNA
19
Q
- Which of the following is NOT required for mammalian cell polyadenylation of pre-mRNA?
A. PAP B. CPSF C. PABII D. RNA polymerase I E. RNA polymerase II
A
**DNA Polymerase I (if that is an option)
D. RNA polymerase I
20
Q
- Poly(A) polymerase can be found in the _________ of a cell.
A. nucleus B. cytoplasm C. lysosomes D. nucleus and cytoplasm E. nucleus and lysosomes
A
D. nucleus and cytoplasm
21
Q
- The capping substance for eukaryotic mRNAs is ______________.
A
7-methylguanosine
22
Q
- The 5 cap of mRNA helps protect the mature mRNA from degradation by _____.
A
RNases
23
Q
- _____ RNA is the only type of RNA to get polyadenylated.
A
Messenger
24
Q
- T/F: Uncapped mRNA is much more stable then capped mRNA.
A
False
25
9. T/F: The 5 cap of mRNA has no effect on the translatability of the mRNA.
False
26
10. T/F: Poly(A) tails are added to the 5 end of the mRNA molecule.
False
27
11. Most genes do not encode for a poly(A) tail.
True
28
12. T/F: Pre-mRNA must be cleaved before it is polyadenylated.
True
29
13. T/F: Polyadenylation is required for efficient transport of mRNA out of the nucleus.
True
30
1. Which of the following mammalian cell DNA polymerases is not correctly matched with its function?
A. DNA Pol —priming DNA synthesis
B. DNA Pol —DNA repair
C. DNA Pol —elongation of both strands
D. DNA Pol —ligation of DNA strands
E. DNA Pol —forms phosphodiester bonds
D. DNA Pol —ligation of DNA strands
31
2. Which of the following agents causes DNA damage by forming pyrimidine dimers?
A. X-rays
B. gamma radiation
C. UV radiation
D. alkylating agents
E. ethidium bromide
C. UV radiation
32
3. The ____ strand in DNA replication is synthesized discontinuously.
lagging
33
4. ______ refers to the fact that once DNA polymerase holoenzyme starts replicating DNA it remains bound to the DNA template and catalyzes the addition of several nucleotides to the growing strand.
Processivity
34
5. The enzyme responsible for bacterial DNA replication is ____.
DNA Polymerase III
35
6. In most organisms the direction of DNA replication is _____.
bidirectional
36
7. Enzymes that introduce transient single-stranded or double-stranded breaks in DNA are called ____.
DNA topoisomerase
37
8. The process by which electrophiles, such as ethylmethane sulfonate, add carbon containing groups to DNA is called ____.
alkylation
38
9. E. coli Base Excision repair starts with the enzyme _____ clipping out the damaged base and leaving an apurinic or apyrimidinic site.
DNA glycosylase
39
10. By ____ the adenine found in the sequence GATC on the parental DNA strand, E. coli is able to differentiate the parental strand from the mismatched base pairs in the daughter strand.
methylating
40
11. T/F: E. coli DNA Polymerase III does not have perfect fidelity. This is a beneficial characteristic since mutations help the cell adapt to changing environments.
True
41
12. T/F: DNA photolyase in human cells is involved in breaking pyrimidine dimers formed by UV radiation.
False
42
13. T/F: Double stranded DNA breaks caused by ionizing radiation are the most dangerous type of DNA damage due to repair by homologous recombination.
False
43
14. A primosome can be defined as
A. the enzyme that synthesizes primers fro M13 phage replication.
B. a collection of proteins needed for synthesis of primers for replicating DNA.
C. a collection of RNA primers.
D. a DNA molecule that is primed and ready for replication.
E. an area of DNA near the replication fork waiting to be replicated.
B. a collection of proteins needed for synthesis of primers for replicating DNA.
44
15. Put the following steps of E. coli primosome assembly in the correct order.
(1) Primase binds.
(2) DnaA binds to oriC at dnaA boxes.
(3) DnaB binds to the open complex.
(4) DnaA, RNA polymerase, and HU protein melt the DNA.
A. 2, 4, 1, 3
B. 2, 4, 3, 1
C. 4, 1, 2, 3
D. 4, 2, 1, 3
E. 3, 4, 1, 2
B. 2, 4, 3, 1
45
16. DNA polymerase III is a highly ____ enzyme, meaning once it binds DNA it catalyzes the addition of several hundreds of nucleotides without falling of the DNA.
processive
46
17. The DNA polymerase III core plus the ___ is needed to replicate DNA processively.
beta-clamp
47
18. Loading of the beta-clamp onto DNA by the clamp loader is an ___-dependent process.
ATP
48
What are the four main functions of Caps?
1. Protection from degradation
2. Improvement of translatability
3. Facilitate transport of RNA (mRNA out of the nucleus)
4. Proper splicing of pre-mRNA
49
What is polyadenylation? What evidence exists that the poly(A) is added post-transcriptionally?
Option 1: It is the modification of the 3' end; addition of AMP chain
Option 2: addition of a poly (A) tail to the 3' end of an mRNA
Evidence: 250 nt sequence
50
What are the two main functions of polyadenylation?
1. Protection of RNA
| 2. Translatatbility of RNA
51
What are the main steps in polyadenylation?
1. Cleavage of mRNA
2. 3' end of mRNA is polyadenylated:
Requires RNA binding proteins CPSF (binds to AAUAAA signal) and CSTF (binds to U/GU rich region)' PAP enzyme is needed for polyadenylation at 3' end; the PABP stimulates the activity of the PAP; and the XRN-2 degrades the leftover mRNA tail
52
What enzymes are needed for polyadenylation to occur?
Poly (A) polymerase
53
What are the differences between the Cap1, Cap2, and Cap3 products and in which organism(s) are each respective Caps found?
```
Cap 1 :
-found in eukaryotic and viral RNAs
-Products - 1) methylation of N7 of terminal guanine 2) methylation of 2'-hydroxyl of penultimate nucleotide
Cap2:
-only in eukaryotic RNA
-Product - next nucleotide (y) methylated in a repeat od methylation of 2'-OH penulti
Cap0:
-only found in some viral RNA
-no 2'-O methylated nucleotides
```
54
What are the “five phases” of an intron life-span?
1. Genomic intron
2. Transcribed intron
3. Spliced intron
4. Excised intron
5. EJC harboring transcript
55
What is the exon-junction complex?
A complex of mRNA with proteins just added upstream of exon-exon junctions at the time of splicing. The proteins facilitate the transport of mRNP out of the nucleus and into the cytoplasm.
56
What are the basic splicing signals?
GU - first 2 bases of introns
| AG - last 2 introns
57
Describe the simplified mechanism of RNA splicing.
1) 2'-hydroxyl group of an adenine nucleotide within an intron attacks phosphodiester bond that links the first exon and intron; yields - free exon 1 and lariat-shaped intron
2) The free 3'-OH group on exon 1 attacks the phosphodiester bond between exon 2 and intron; yields - spliced exon1/exon2 product
58
Describe the spliceosome cycle.
1. Assembly 2. Function 3. Disassembly
A commitment complex is formed when U1 binds to splicing substrate. U2 joins the complex using the ATP, followed by others. U6 disassociates from U4 and displaces U1 at 5' splice site.
59
What is the role of the CTD of RNA polymerase in splicing?
1) Stimulates splicing of substrates that use exon definition
2) binds to splicing factors and could assemble the factors at the end of exons to set them off for splicing.
60
List the different types of alternative splicing.
- Begin transcripts at alternative promoters
- Exon skipping - some exons may be ignored resulting in deletion of exon
- Alternative acceptor site - alternative 5'-splice site can lead to inclusion or deletion of part of an exon
- Alternative donor site - alternative 3'-splice site can lead to inclusion or deletion of part of an exon
- Intron retention - intron retained in mRNA may not be recognized as an intron
- Polyadenylation
61
How would you test for exonic splicing silencers?
1) Computational method: looking for sequences enriched in pseudoexons versus real exons
OR
2) Reporter construct: looks directly for sequences that inhibit splicing
62
Describe the mechanism for self-splicing RNAs ?
Self‑splicing occurs by a phosphoester transfer mechanism. The 3'‑OH of the guanine nucleotide is the nucleophile that attacks and joins to the 5' phosphate of the first nucleotide of the intron.
63
What is a Klenow fragment?
Fragment of DNA Polymerase I that was made by cleaving with a protease, lacks 5'->3' exonuclease activity of parent enzyme.
64
How is fidelity of replication maintained?
The fidelity of DNA replication relies on:
- nucleotide selectivity of replicative DNA polymerase
- exonucleolytic proofreading,
- postreplicative DNA mismatch repair (MMR)
65
What is DNA gyrase? What reaction does it catalyze?
A topoisomerase
an essential bacterial enzyme that catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA
66
What are topoisomerases and what do they do?
An enzyme that changes a DNA's superhelical form or topology.
Type I - transient single-strand breaks into substrate DNA's
Type II - transient double-strand breaks into substrate DNA's
67
What are the major types of DNA damage?
1) Base modification by alkylating agents
| 2) Pyrimidine dimers caused by UV radiation
68
Describe one mechanism of direct DNA repair.
UV radiation damage repair can be undone directly by a DNA photolyase which uses energy from near-UV to blue light to break bonds holding 2 pyrimidines together.
69
What is the difference between BER and NER? How does each work?
BER - acts on subtle base damage using DNA glycosylase
DNA glycosylase extrudes and removes the damaged base, leaving an apurinic and apyrimidinic site on the bottom of the strand. DNA repair enzymes remove the remaining deoxyribose phosphate and replace it with a normal nucleotide.
NER - handles bulky damage that distorts the DNA helix without the use of DNA glycosylase
The uvrABC excinuclease makes cuts on both side of bulky damage and removes oligonucleotide. DNA I polymerase fills in the missing strands and DNA ligase seals the nicks for both methods.
70
How are double-stranded breaks in eukaryotes repaired?
Nonhomologous end-joining OR
| Homologous recombination
71
What is error-prone bypass and how does it work?
A mechanism cells use to replicate DNA with pyrimidine dimers or noncoding bases.
In E.coli, the SOS response is induced causing DNA to replicate even though the damage region can't be read correctly resulting in errors in the newly made DNA.
72
Outline the process of initiation of DNA replication in prokaryotes.
Initiator proteins bind to the ori-c creating a complex that helps to initially separate DNA.
DNA helicase unwinds DNA by breaking the hydrogen bonds between the complementary strands separating DNA into two template strands. Unwinding and synthesis continue in both directions creating 2 replication forks.
73
What are the different mechanisms by which pyrimidine dimers can be corrected?
Photoreactivation
| Nucleotide excision repair
74
How can alkylation be corrected?
Method of O6-methylguanine methyltransferase:
O6 alkylations on guanine residues can be directly reversed by the suicide enzyme O6-methylguanine methyltransferase, which accepts the alkyl group onto one of its amino acids.
75
Outline the process of DNA replication synthesis (after initiation) in prokaryotes.
During elongation, the prokaryotic RNA polymerase tracks along the DNA template, synthesizes mRNA in the 5′ to 3′ direction, and unwinds and rewinds the DNA as it is read.
the addition of nucleotides to the 3′-end of a growing RNA chain during transcription
76
Describe the process of DNA replication termination in prokaryotes.
occurs when the two forks meet and fuse at the terminus region, creating two separate double‐stranded DNA molecules.
77
List the E.coli polymerases and their major functions as well as enzymatic activities.
DNA Polymerase I - process Okazaki fragments & fills in gaps during DNA repair
DNA Polymerase II - proofreading and editing (mainly on lagging strand)
DNA Polymerase III - replication of bacterial DNA
78
What is priming and how does the primosome assembly at OriC occurs in DNA replication in E. coli ?
Priming - initiation step in DNA replication
Primosome assembly at the origin of replication, oriC, occurs as follows:
1) DnaA binds to oriC at sites called dnaA boxes and cooperates with RNA polymerase and HU protein in melting a DNA region adjacent to the leftmost dnaA box.
2) DnaB then binds to the open complex and facilitates binding of the primase to complete the primosome.
3) The primosome remains with the replisome, repeatedly priming Okazaki fragment synthesis, at least on the lagging strand. DnaB also has a helicase activity that unwinds the DNA as the replisome progresses.
79
Describe the structure, i.e. subunits of pol III holoenzyme and describe briefly the function of each subunit.
beta-subunit - sliding clamps structure (processivity factor)
y (gamma) complex - loads/unloads beta clamp
Core enzyme (alpha, epsilon, theta) - polymerization and proofreading
t (tau) subunit - dimerizes core complex
pol III holoenzyme - active form of DNA polymerase III; dimer with 10 polypeptide subunits
80
Define processivity of replication and which subunit of pol III holoenzyme carries out this function in DNA replication in E. coli.
Processivity - the ability of an enzyme to remain bound to one or more of its substrates without falling off and having to reinitiate
Beta-subunit
81
Describe the role(s) of γ complex in DNA replication in E. coli and demonstrate one of its functions experimentally.
y (gamma) complex - loads/unloads beta clamp
O'Donnell (1991) clamp loader function experiment demonstrated that y complex itself is not the agent that provides processivity; y complex could act catalytically to the core polymerase that makes it processive.
82
How the lagging strand is synthesized in E. coli DNA replication?
synthesized as discrete, short fragments (Okazaki fragments) backward from the direction of replication. discontinuously.
clamped polymerase gets to end of Okazaki->Beta-clamp loses affinity for core, binds to gamma complex (clamp loader), unloads polymerase, polymerase binds another primer sequence, clamp loads back on, polymerase continues
83
Demonstrate experimentally that sliding clamp performs its function only on circular double-stranded DNA.
O'Donnell and Kuriyan used x-ray crystallopgraphy to study the b-clamp structure which forms a ring that can fit around DNA. This fit into the rationale that a b clamp could remain bound to circular DNA versus a linear one - example a ring over a string it can readily fall off but not if the string is circular.
84
How the problem of filling of DNA gaps at the ends is solved in the termination step in DNA replication in eukaryotes.
Describe/diagrammatically the steps involved, i.e. what happens at the ends of eukaryotic chromosomes.
Euks:
linear, polymerase cant go 3-->5
so no 3' end upstream for polymerase to come back and complete
DNA would get shorter and shorter
added repeat sequences by enzyme TELOMERASE
two components: 1. protein 2. RNA
after replication, one strand has free 3' extension, RNA forms complementary base pairing with overhang, 3 bp used to make TTG, Primase can add, DNA polymerase fills it in, RNA primer digested away, 3' overhang longer
85
What is epigenetics and what are the three major mechanisms that impact epigenetics?
Epigenetics - study of changes in the regulation of gene activity and expression not dependent on DNA sequence
1. DNA methylation
2. RNA interference
3. Histone modification
86
Briefly describe the two major mechanisms of RNA interference.
1) Post-transcriptional silencing targets mRNA for degradation or blockas translation.
2) Transcriptional silencing targets transcription of the gene directly.
87
How does DNA methylation impact gene expression? What are the targets for DNA methylation?
the addition of a methyl (CH3) group to DNA, thereby often modifying the function of the genes and affecting gene expression
5-methylcytosine
88
What is an imprinted gene? Explain by providing an example.
the ability of a gene to be expressed depends upon the sex of the parent who passed on the gene
Prader-Willi syndrome - Inheritance of a deleted chromosome 15 from the father
89
Provide a brief description of the relationship between DNA methylation and cancer
Cancer cells contain altered DNA methylation patterns.
Hypomethylation - can lead to perturbations in gene expression or genomic instability.
Hypermethylation - may promote widespread changes in gene expression patternsthrough different mechanisms.
90
In terms of histone modifications, what are cis-effects, trans-effects, and histone replacement?
cis-effects: alter nucleosomal contacts and spacing
trans-effects: altered histone - non histone protein associations
Histone replacement - replacement of canonical histone by a noncanonical variant
91
What are chromodomain and bromodomains?
chromodomain - a conserved region found in proteins involved in the formation of chromatin.
bromodomain - protein domain that binds specifically to acetylated lysine residues on other proteins, such as histones.
92
The basic steps of translation as outlined.
1. Initiation
2. Elongation
3. Termination
93
Where does GTP hydrolysis occur?
In all 3 steps.
Initiation: on IF-2
Elongation: EF-Tu, EF-G
Termination: RF-3
94
The various antibiotics listed and their effects.
Streptomycin - interferes with the initiation of protein synthesis
Tetracyclines - interacts with 30S ribosomal subunits blocking access of the aminoacyl-tRNA
Puromycin - inhibits further elongation
Chloramphenicol - inhibits peptidyltransferase; high levels may inhibit mitochondrial protein synthesis
Clindamycin and Erythromycin - inhibits translocation
95
Compare and contrast how prokaryotes and eukaryotes start the translation.
Prokaryotes:
- Only 3 initiation factors required: IF1, IF2, IF3
- Many start sites and SD sequence all along mRNA
- Initiation codon is AUG, sometimes GUG or UUG
Eukaryotic:
- only cytoplasmic ribosomes can initiate translation
- Two types of translation initiated mechanisms - cap dependent, and cap independent
- Has only one transcription start site
- SD sequence is absent in mRNA
- 9 initiation factors
