1
Q
Transcription + Translation =
A
Transcription + Translation = Gene Expression
2
Q
What does the process of transcription and translation together lead to?
A
The process of transcription and translation together
refers to GENE EXPRESSION
3
Q
How long are typical miRNA and siRNA molecules?
A
microRNA and small-interferingRNA are typically 22-25 nucleotides long
4
Q
How does control of gene expression with RNA interference occur?
A
Usually controlled by dsRNA molecules that are used in gene silencing, preventing expression of certain genes in a cell.
5
Q
Why might have RNAi evolved?
A
RNAi may have evolved as an immune system for the genome as a defence mechanism against viruses.
6
Q
Where do siRNA and miRNA interfere?
A
siRNA and miRNA both interfere following transcription or post-transcription mutation
7
Q
What are two types of RNAi that occur after transcription?
A
- The miRNA is destroyed before it is translated into protein
- Inhibits translation and protien is therefore not made
8
Q
What are the precursors of miRNAs called and what are they encoded by?
A
The precursors of miRNAs, called primary miRNA (pri-miRNA), are encoded by the
genome.
- The relevant genomic regions are transcribed by RNA Pol II.
9
Q
Where is Pri-miRNA cleaved and by what mechanism?
A
In the nucleus, Pri-miRNA is cleaved by Drosha (RNAse III enzyme) into pre-miRNA,
a stem-loop structure
10
Q
What occurs in the cytosol with the Dicer enzyme?
A
In the cytosol, Dicer cleaves the pre-miRNA into 19–25 nucleotide miRNA:miRNA
duplex with no stem-loop
11
Q
Why does RNA make a structure of its own?
A
It can do this due to the presence of inverted repeats
12
Q
How does RNAi work with siRNA?
A
- Passenger strand is cleaved and incorporated to siRISC
- Complementary binding of the guide strand to mRNA
- mRNA cleavage
13
Q
How does RNAi work with miRNA?
A
- Passenger strand is discarded and the rest is brought into miRISC
- Incomplete complementary binding of the guide strand to target mRNA
- Translated repression, mRNA degradation and cleavage
14
Q
Differences between miRNA and siRNA?
A
miRNA has inverted repeats and siRNA has external RNA with 2 different genes in the genome
15
Q
miRNA has…?
A
imperfect base pairing
16
Q
siRNA has…?
A
perfect base pairing
17
Q
What is siRNA like before dicer processing?
A
Double-stranded RNA that
contains up to 100
nucleotides
18
Q
What is the actual siRNA structure?
A
21-23 nucleotide RNA
duplex with 2 nucleotides
3‘overhang
19
Q
Complementary siRNA?
A
Fully complementary to mRNA
20
Q
mRNA target of siRNA?
A
One target
21
Q
Mechanism of gene regulation for siRNA?
A
Endonucleolytic cleavage of mRNA
22
Q
miRNA prior to dicer processing?
A
Precursor miRNA (pre-miRNA) that contains 70-100 nucleotides with interspersed mismatches and hairpin structure
23
Q
Structure of miRNA?
A
19-25 nucleotide RNA duplex
usually with 2 nucleotides
3’overhang
24
Q
miRNA complementary?
A
Partially complementary to mRNA, typically targeting the 3’
untranslated region of mRNA
25
miRNA mRNA target?
Multiple (could be over 100 at the same time)
26
Mechanism of gene regulation for miRNA?
- Translational repression
- Degradation of mRNA
- Endonucleolytic cleavage of mRNA (when there is a high level of complementary between miRNA and mRNA)
27
Transcriptional gene silencing?
RNAi during transcription process
- condenses chromatin to suppress transcription, mRNA is not made
28
siRNA interference works how?
Degrade mRNA by cleavage
29
miRNA interference works how?
lead to inhibition of translation and/or mRNA degradation
30
transcriptional siRNA mechanism?
brings about methylation of histone proteins or DNA, inhibiting transcription
31
What is another role of RNAi?
Another role of RNAi is to direct localized repressive
chromatin formation.
32
Where are siRNA duplexes loaded?
Small RNA (siRNA) duplexes are loaded into a nuclear
form of RISC (cytoplasm) , called the RNA-induced transcriptional silencing (RITS) (nucleus) complex.
33
What is RITS and what does it do?
Similar to RISC, RITS are effector complexes that are
targeted to homologous sequences by base-pairing
interactions involving the guide strand of the small RNA.
34
What does RITS mediate?
RITS mediates gene silencing via heterochromatin
formation.
35
When are we unable to do transcription?
Transcription cannot occur when the nucleosomes are formed (complex of DNA and histones)
36
What do changes in chromatin structure affect?
Changes in chromatin structure affect the expression of eukaryotic genes
37
What is heterochromatin?
Heterochromatin is the condensed inactive form of
chromatin.
38
What is euchromatin?
Euchromatin is the open and active form of chromatin.
39
What happens when the chromatin structure is open?
When the chromatin structure is open, transcription
factors access the DNA and initiate transcription.
40
How does histone modification occur?
Histone modification occurs at multiple sites by acetylation, methylation and phosphorylation
41
How does the RITS work?
siRNAs in RITS can base-pair with RNAs that are transcribed from their target DNA.
- RITS acts as a recruiter of modifying enzymes to specific DNA regions.
- Enzymes, now catalyse the methylation of histone tails or cytosine bases in DNA
42
How many of our genes are thought to be regulated by RNAi?
Approximately, 30% of human genes thought to be
regulated by RNAi; thousands of genes code for miRNAs.
43
What is RNAi important for?
Very important for regulating gene expression during
embryo development.
44
Why is RNAi important for research?
Important research tool for ‘knocking out’ (silencing)
particular genes.
45
What might RNAi lead to?
Lead to major advances in the treatment of human diseases.
46
Treatment of cancer with RNAi approach?
Combination approach of targeted delivery of siRNA and chemo drugs to the cytoplasm of a cancer cell.
47
What happens to the siRNA-containing nanoparticle?
The siRNA-containing nanoparticle is taken into the cell by endocytosis. The
nanoparticle unpacks and the siRNA is released.
48
What does a continuous sequence of nucleotides encode?
Continuous sequence of nucleotides encodes a continuous sequence
of amino acids.
49
What is the number of nucleotides proportional to?
Number of nucleotides in the gene is proportional to the number on amino acids in the protein.
50
if we have 24 bp, how many codons and aas do we have?
24 bp
8 codons
8 amino acids
51
What is different for the relationship for eukaryotes?
This process gets messed up in eukaryotes due to the removal of introns because we splice them out (less amino acids total)
52
Colinearity?
With collinearity, the number of nucleotides in the gene is proportional to the number of amino acids in the protein
53
Where are pre-mRNA molecules made?
Eukaryotes and prokaryotes make mRNA
- Eukayotes just have pre-mRNA and snRNA
54
What does a gene include?
A gene includes DNA sequence that codes for all exons,
introns, and those sequences at the beginning and end of
the RNA that are not translated into a protein.
55
Where do transcription and translation start?
Transcription and translation have different start and stop sites (translation sites lie inside the transcribed region)
56
Difference in genome organization between prokaryotes and eukaryotes?
Prokaryotes: transcription and translation occur in the same compartment at the same time
Eukaryotes: transcription and mRNA processing occurs in the nucleus, translation occurs in the cytoplasm
57
What type of mRNA can be made in prokaryotes?
They can make polycistronic mRNA, with multiple start sets controlled by one promotor (all new used too make the same product)
58
What is polycistronic mRNA?
This is when multiple genes are encoded in one mRNA piece.
59
What happens when we need multiple genes transcribed in eukaryotes?
There are 4 different chromosomes all transcribed individually and translated individually.
60
What is an intron?
The intron is the part that does not code for parts of the protein
61
What is an exon?
Exons are sequences we want to have in the final protein sequence.
62
What is the splicing mechanism of nuclear pre-mRNA?
They use spliceosomal mechanism to get protein-encoding genes in the nucleus of eukaryotes
63
What is the order of the different components of a eukaryotic gene?
1. TATA box
2. transcription start site
3. 5' UTR
4. translation start
5. translation stop
6. 3' UTR
7. polyA consensus
8. transcription stop
64
Where are prokaryotic protein coding genes found?
usually found in a contiguous
array in the DNA called an Operon.
65
How do operons operate?
Operon operates as a unit
utilizing a single transcription start site for multiple genes.
66
How does eukaryotic protein-coding genes work?
each gene is transcribed from its own start site to yield a pre-mRNA that is processed into a
functional mRNA encoding a single protein.
67
Prokaryotic genes- introns?
In prokaryotic DNA genes contain little to no noncoding gaps (introns) and the DNA is transcribed directly into colinear mRNA, which then is translated into protein while the mRNA is still being produced
68
What is the function of the 5' cap?
Facilitates binding fo ribosome to the 5' end of mRNA, increases mRNA stability and enhances RNA splicing
69
What is the function of the 3' cleavage and addition of the polyA tail?
Increases stability of the mRNA, aids in export of mRNA from the nucleus, and facilitates binding of ribosome to mRNA
70
What is the function of RNA splicing?
Removes noncoding introns from the pre-mRNA, facilitates export of mRNA to cytoplasm, allows for multiple proteins to be produced through alternative splicing
71
When are mRNAs modified?
Messenger RNAs (mRNAs), which encode the amino acid sequences of proteins, are modified after transcription.
72
What are the three mRNA modifications?
Modifications (inside nucleus):
1) Capping of the 5’ end
2) Polyadenylation of the 3’
3) Splicing (removal) of introns
73
How does 5' capping occur?
A methylated guanine
nucleotide is attached to the
5′-end of the pre-mRNA.
- Methyl groups (CH3) are added
74
How is the methylated guanine nucleotide added?
A methylated guanine (G)
nucleotide is joined to the pre-mRNA by an unusual 5’-5’ linkage involving 3 phosphate groups.
75
Why is modification of the 5' end needed?
Modification is necessary for efficient initiation of translation, transport of mRNA from nucleus, protects mRNA from degradation, and enhances RNA splicing
76
How does polyadenylation occur on the 3' end?
~ 50 to 250 adenine (A) nucleotides are added to the 3′-end of the pre-mRNA
77
How is the site for polyadenylation recognized?
The presence of the cleavage site, consensus sequence and a u-rich sequences are needed for the addition of the polyA tail
78
What is polyadenylation needed for?
Modification is necessary for efficient translation and protects mRNA from
degradation.
79
What does splicing require?
Requires three consensus sequences in the pre-mRNA:
5’ splice site, 3’ splice site and branch point.
80
What are consensus sequences used by?
Consensus sequences are used by the SPLICEOSOME to
recognize and remove introns.
81
What region is known as the intron?
Between the 5' and 3' consensus sequences is the intron
82
How are introns removed?
Introns are removed in the form of a lariat and exons are spliced together by two successive reactions
83
Where does the 5' end of the intron meet?
It meets at the branch point!!!
84
What is the splicosome?
A Ribonucleoprotein complex
[300 proteins and 5 small nuclear RNAs (snRNAs)]
85
What does the spliceosome contain?
Contains five snRNPs (small
nuclear ribonuclear proteins).
-snRNA + protein = snRNP
-U1, U2, U4, U5 and U6
86
What are snRNPs central to?
snRNPs are central to the
activity of the spliceosome.
87
How does the spliceosome assemble?
The snRNAs of U1 and U2 base
pair with the consensus sequence at the 5’ splice site and the branch point site of the pre-mRNA.
88
What are the steps of spliceosome assembly?
1. U1 attaches to 5' splice site
2. U2 attaches to branch point
3. U4,5,6 joins the spliceosome
4. U1 and U4 are released
5. Base pairing between the mRNA sequences and snRNAs hold the spliceosome together
6. The exons are joined together and the intron is released as a lariat
89
You will create double-stranded siRNAs with homology to different regions of the gene. Once incorporated into RISC protein complex, which ones
will effectively silence gene expression?
Transcription start site, start codon, 3' UTR, etc.
NOT INTRON because they have been spliced out already
90
What processes are coupled?
Functional coupling of mRNA transcription and mRNA
processing by RNA polymerase II (Pol II).
91
What is the coupling of transcription and processing mediated by?
Coupling of events are mediated by the ‘tail’ or C-
terminal repeat domain (CTD) of the largest subunit of
Pol II.
92
When are mRNA processing enzymes recruited?
mRNA processing enzymes are recruited to the CTD of
Pol II during transcription.
93
When is CAP added?
CAP is added as soon as the 5’ end of the pre-mRNA emerges from the polymerase.
94
Where are capping enzymes recruited and when?
Capping enzymes are recruited to the C-terminal domain (CTD) of RNA polymerase (Pol) II during the early stages of transcription.
95
What is the role of RNAP II?
RNAP II mediates the functional coupling of transcription to splicing
by directing the nascent pre-mRNA into spliceosome assembly
96
When is the spliceosome formed?
Assembly of the spliceosome
occur co-transcriptionally,
while the RNA polymerase
(Pol II) is still actively
transcribing the template.
97
Components of the spliceosome are added when?
Components of the
spliceosome are recruited to
the RNA while transcription is
occurring.
- Scans along while mRNA is made, cuts introns when they emerge
98
How does the CTD of Pol II interact with proteins?
The CTD of Pol II interacts
directly with splicing proteins
to recruit them to the RNA.
99
Does transcription end at a specific sequence?
Transcription does not end at a specific sequence
100
What does termination require?
Termination requires cleavage of the mRNA at a specific site
101
What does the 5'->3' exonuclease do?
A 5' -> 3' exonuclease degrades the remaining mRNA terminating transcription
102
When does polyadenylation occur and how?
~50-250 adenines are added to the 3' end of the pre-mRNA
103
How are polyadenylation factors recruited?
Polyadenylation factors are
recruited to the CTD of Pol
II.
104
What is the role of Pol II?
Its makes mRNA and also recruits enzymes to mature the mRNA
105
Where is RNA cleaved?
RNA is cleaved at the
Poly(A) 3’ cleavage site.
106
How is the remaining RNA degraded?
Degradation of the
remaining RNA by Rat1
terminates transcription.
107
How is Rat1 loaded?
Rat1 is loaded by the CTD
108
How many protein coding genes are there?
Human genome sequence published in 2001, says that
there are ~20,000 protein-encoding genes
109
How is the proteome more complicated?
The proteome meaning the protein complement of the cell
is a bit more complicated than the genome because a
single gene can give rise to a number of different proteins
110
How many proteins can we produce?
We can produce over 100,000
proteins from 20,000 genes.
111
How can pre-mRNA be processed?
A single pre-mRNA can be processed in different ways to
produce different mRNA molecules thereby translating to different proteins.
112
What are the two different methods for processing pre-mRNA?
Alternative splicing or alternative polyA site
113
Alternative splicing?
Alternative splicing: pre-mRNA can be spliced in different ways.
114
Alternative PolyA site?
Alternative PolyA site: PolyA tail can be added at
different 3’ cleavage sites.
115
What does each mRNA have different?
Each mRNA produced has
a different combination of
exons.
116
What does each mRNA produce?
Each mRNA when
translated produces a
different protein (isoforms
of protein).
117
What are the 4 different methods for alternative splicing?
1. exon skipped
2. intron retention
3. alternative 5' or 3' splice site
4. mutually exclusive exons
118
exon skipped?
uses the 3' cleavage site that ends up skipping an exon
119
intron retention?
doesnt splice out an intron
120
alternative 5' or 3' splice sites?
A splice site in the middle of an exon would be used, making less of that exon available
121
mutually exclusive exons?
either cannot have orange or green - both cannot be present
122
pre-mRNA contains..?
Pre-mRNA contains
multiple 3’ cleavage sites.W
123
What determines the length of the mRNA transcript?
The 3’ cleavage site used
determines the length of
the mRNA transcript.
124
What does each mRNA produce when translated?
Each mRNA when
translated produces
similar proteins of
different size.
125
How is alternative processing regulated?
Switch between the production of a functional or nonfunctional protein.
Ex. Drosophila sex determination (non functional is male phenotype and functional is female). B exon is spliced out to be functional
126
When might different forms of a protein be made?
Different forms of a protein may be produced in
different cell types.
127
What might a genetic disease arise from?
Many genetic disease arise from mutations that affect pre-mRNA splicing.
128
What processes might genetic diseases inhibit/interfere with?
o Affect use of splice sites
o Affect the splicing machinery
o Affect regulators of alternative splicing
129
How many mutations alter pre-mRNA splicing?
About 15% of all single nucleotide mutations (point mutations) that cause
diseases alter pre-mRNA splicing.
130
What might mutations affecting splice sites do?
- disrupt the consensus sequence so that the spliceosome can no longer recognize the 5’ or 3’ splice site.
- create a new 5’ or 3’ splice site.
- initiate usage of an existing cryptic 5’ or 3’ splice site
131
What might mutations lead to? What can this result in?
Mutations can lead to exon skipping (complete or partial) or intron retention (complete or partial).
- This result in gene loss of function either by generating a nonfunctional protein or by altering the stability of the
mRNA (mRNA is degraded and no protein is made).
132
What does splicing require?
Requires three consensus sequences in the pre-mRNA:
5’ splice site, 3’ splice site and branch point.
133
What does the spliceosome use?
Consensus sequences are used by the SPLICEOSOME to
recognize and remove introns.
134
What is the spliceosome?
A Ribonucleoprotein complex
[300 proteins and 5 small nuclear RNAs (snRNAs)].
135
What does the spliceosome contain?
Contains five snRNPs (small
nuclear ribonuclear proteins).
-snRNA + protein = snRNP
-U1, U2, U4, U5 and U6
136
What are snRNPs central to?
snRNPs are central to the
activity of the spliceosome.
137
What are the 2 things that mutations to splice sites causes?
Mutations affecting splice sites can lead to exon skipping or intron retention (occurs less frequently)
138
What might mutations initiate?
Mutations may initiate use of a cryptic splice site (a sequence not normally used for splicing) or create a new splice site
139
Does a gene always match its RNA product? Why?
Occasionally a gene is found with a sequence of
nucleotides that does not exactly match that of its RNA
product.
- Editing alters the coding information of the of mRNA
transcripts.
140
What are the 2 methods of RNA editing?
1) Substitution Editing (base conversion)
2) Insertion Editing
141
Substitution editing?
Chemical alteration of individual nucleotides by specific enzymes.
- Example C is converted to U.
142
How does addition of U nucleotides occur?
Addition of U nucleotides
occurs by cleavage of the
mRNA, insertion of the U
nucleotide, and ligation of
the ends.
143
How are insertion reactions catalyzed?
The reactions are catalyzed
by a complex of enzymes
under the direction of
guide RNA (gRNA).
144
How do gRNAs base-pair?
gRNAs base-pair as best
they can with the mRNA to
be edited and serve as
a template for the addition
of nucleotides.
145
What is the role of guide RNA?
guide RNA adds nucleotides to the pre-mRNA that were not encoded by the DNA
146
How are U's added?
U's are added to base pair with the A's that have been added.
147
When is RNA processed in eukaryotes?
Eukaryotic RNA is processed before export to the cytoplasm and translation.
148
What is added to the pre-mRNA?
A modified nucleotide is added to 5’ end (capping) and a polyA tail is added to the
3’end (polyadenylation) of the pre-mRNA.
149
What is removed from the pre-mRNA and how does this happen?
Introns (noncoding sequences) are removed from pre-mRNA and exons (coding sequences) are spliced together by two transesterification reactions to generate the mature mRNA.
- Splicing takes place in the large ribonucleoprotein complex, the spliceosome.
150
What is alternative processing and what does it produce?
Alternative processing of a pre-mRNA produces different types of mRNA, resulting in
the production of different types of proteins from a single gene. Increases protein
diversity.
151
RNA editing alters what and how?
RNA editing can also alter the protein product of a gene by changing the nucleotide
sequence of the mRNA. Nucleotides are substituted, deleted or inserted.
BIOL2030
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