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RNA (new) Flashcards

1
Q

What enzyme do we need to regulate in Transcription of Eukaryotes

A

RNA polymerase II

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2
Q

RNA polymerase II

A
  • Cannot initiate transcription
  • Only elongation of existing RNA strand
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3
Q

Initiation complex of Euk. Transcription

A
  • TFII A/B/D/E/F/H
  • RNA polymerase II
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4
Q

What regulates Initiation of Euk. Transcription

A

Mediator Complex
- Enhance or block transcription initiation by receiving signals from activators or repressors.

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5
Q

Spacer DNA

A
  • Long undefined sequences of DNA (100s/1000s nucleotides)
  • Between the regulatory sequences
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6
Q

What is the main way Euk. Transcription is Regulated

A

Packing/Unpacking of heterochromatin and Euchromatin
since in packed DNA TATA box is not available

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7
Q

What causes unpacking of DNA to euchromatin

A

Prime activator proteins which recruit co-activators

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8
Q

2 Pathways after Prime activator

A
  • Chromatin Remodeling Complex
    OR
  • Histone Modifying Enzyme
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9
Q

Co-Activator Chromatin Remodeling Complex

A
  • Recruited by Activator protein
  • Surrounds large area of DNA (4/5 nucleosomes and loosens them
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10
Q

Co-Activator Histone Modifying Enzyme

A
  • Recruited by Activator protein
  • Covalently modifies Histones by histone acetylation
  • Positive charge is lost weakening interaction with DNA and loosening it
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11
Q

What happens when DNA unfolds and Core promoter is seen

A

1) Activator protein binds to enhancer sequence
2) Activator protein recruits Histone acetyltransferase acetylating lysine residues
3) Histone Kinase also recruited phosphorylating serine on histones for more repulsion

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12
Q

Histone Code

A

Pattern of modifications happening to loosen DNA by histone kinase and histone acetyltransferase

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13
Q

What does Histone code recruit and what does it do

A

Chromatin Remodeling complex
- Stabilizes the less condensed chromatin, keeping the DNA accessible.

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14
Q

TFIID role in Histone Code

A

TFIID also binds the histone code as well as core promoter
So it can engage the initiation of Transcription

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15
Q

Ways repressor proteins can act

A
  • Block binding of Activator
  • Binds to silencer sequence and competes with bound activator
  • Repressor and activator compete for mediator complex
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16
Q

Co-repressors

A
  • Chromatin remodeling complex
  • Histone deacetylase & Histone methyltransferase
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17
Q

Specific Transcription factors

A
  • Help regulate gene expression by binding to specific DNA sequences called Response Elements
  • Usually Dimeric
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18
Q

Regulation of Specific Transcription factors

A
  • Protein Synthesis (under transcriptional control itself)
  • Ligand binding (Nuclear-R)
  • Covalent Mod. (undoable)
  • Dimerization
  • Unmasking (removing inh.)
  • Stimulation of Nuclear Entry
  • Release from membrane (because they are anchored)
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19
Q

Regulation of Euk. Gene expression by UTR binding

A
  • RNA Binding proteins (RBP)
  • Bind UTR regions
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20
Q

UTR binding example

A

IRE & IREBP in iron starvation

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21
Q

IRE & IREBP in Iron Starvation

A
  • Cytosolic Aconitase (IREBP)
  • Binds IRE of 5’ UTR of mRNA coding Ferritin, and stabilizes 3’ end for transferrin-R
  • Represses translation of mRNA strand of Ferritin
  • No Ferritin so less storage, more Transferrin-R
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22
Q

IRE & IREBP in Iron Excess

A
  • Iron binds Cytosolic Aconitase (IREBP) bound to IRE on mRNA
  • Aconitase removed and no more repression
  • Ferritin Made
  • Aconitase off of 3’ exposes poly-A and causes instability
  • No Transferrin-R made
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23
Q

What transcribes miRNA and how many are there

A

RNA Polymerase II
(500-2000 genes across different species)

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24
Q

miRNA main functions summary

A

Inhibitory
- Cleavage of mRNA into 2 pieces
- Destabilization of mRNA by shortening poly-A tail
- Less efficient translation of mRNA to proteins by ribosomes

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25
Biogenesis of Pri-miRNA (primary)
- Synthesized in Nucleus from microRNA genes - Introns of protein coding genes or non coding RNA genes - Almost all synthesized by RNA polymerase II
26
Structure of Pri-miRNA
- Self-complementarity (fold back on themselves) - 35bp (ds) stem and single stranded loop (not strict pairing) - 5' cap structure and 3' poly-A tail
27
Pri-miRNA Motifs
- Basal UG motif - Flanking CNNC motif - Mismatched GHG motif - Apical UGU motif
28
Biogenesis of Pre-miRNA
- From Pri-mRNA - By Microprocessor (heterotrimeric complex) - Exported to cytoplasm by Exportin5 and GTPase
29
Microprocessor
Primary to Pre-mRNA - Enzyme drosha (ribonuclease III) cuts pri-mRNA at 22nd nucleotide of ssloop - 2 Proteins: DGCR8
30
Biogenesis of miRNA
- By heterotrimeric complex from pre-miRNA - Dicer enzyme (ribonuclease III) + TARBP proteins - Loop is lost and dsRNA formed
31
miRNA activation
- Binding to Ago protein using guide strand - Forms RNA induced silencing complex (RISC) - Other strand (passenger) is degraded
32
How does Ago protein decide which strand to chose as guide from mi-RNA
Prefers one with A or U at 5'end or less stable 5'end
33
RNA editing - miRNA formation modification
- ADAR enzymes catalyze deamination of Adenine to Inosine within the pri-mRNA transcript - Inosine is structurally similar to guanine and is read as guanine by machinery - Causes either Destabilization and degradation of pri-mRNA or an edited mature miRNA with altered specificity
34
What does RNA editing + Alternative cleavage make?
IsomiRs
35
What is edited more frequently RNA transcripts of miRNA genes or protein coding genes?
miRNA since they have a regulatory role
36
Mitrons
- Form from introns of protein coding genes - When intron is spliced it escapes degradation and remains in nucleus - Debranched and forms pre-miRNA
37
Seed region
- Small part of miRNA sequence that is important for finding its target mRNA - First base very important for its specificity: usually Adenine
38
Where does miRNA bind mRNA
At 3' UTR
39
TNRC6 outcomes
- TNRC6 brings proteins to break down mRNA (main way) - TNRC6 can also stop its translation to protein without degradation (less efficient)
40
miRNA vs siRNA
- miRNA: reduce expression of target gene - siRNA: completely eliminate the expression of a target gene
41
DNA methylation
- By SAM - Changes activity of DNA segment without changing the sequence - Usually in CpG dinucleotides (5th carbon of c then g on backbone) - Silences genes
42
Importance of Cytosine deamination in methylation
- Usually C deamination forms Uracil - But, methylated C deamination forms Thymine - This is one of the most frequent mutations
43
Pioneer TFs
Weirdly prefer methylated sites on DNA rather than the usual TFs that prefer unmethylated
44
Cassette Exon
Can be removed with introns or remain as part of the mRNA
45
Alternative 3'/5' splicing
Exon is longer or shorter at the 3'/5' splice site
46
Mutually exclusive exons
2 exons next to each other with an intron in between, but only 1 can remain in the final mRNA so 1 has to be spliced out
47
Alternative Promoter
- More than one promoter on a strand - If first promoter is used whole thing is transcribed - If second promoter is used the previous part is not incorporated in the pre-mRNA
48
How are some introns spliced and some not in other cells?
We have Negative & Positive control with Splicing repressors and Splicing activators which binds intron splice sites
49
Alternative Poly-A site
- Resting B Cell: Low Cstf so weak poly-A site not recognized (membrane bound IgM) - Active B Cell: High Cstf recognizes weak poly-A site and early cleavage (free IgM)
50
mRNA degradation methods
- Poly-A tail removal/shortening - Decapping (allows exonucleases to act)
51
RNA editing
Example that mRNA coding for ApoB-100 has base CAA - In liver: No RNA editing so full mRNA translated - In intestines: RNA editing by deamination and C forms U. (UAA) Early stop codon and truncated protein formed ApoB-48
52
Non-Sequence Specific Approach
Measuring overall gene expression in a sample 1) mRNA reverse transcribed to cDNA (dsDNA) 2) SYBR Green dye used to bind cDNA 3) Fluorescence only when bound to cDNA 4) PCR amplifies cDNA making more and hence more fluorescence detected
53
Advantages of Non-Sequence Specific Approach
- The SYBR Green dye can be used for any genes being studied - It is a non-specific DNA binding dye
54
Disadvantages of Non-Sequence Specific Approach
- Lack of specificity may lead to detection of non-specific amplification products - Primers can form complex with dye giving signal (false signal)
55
Probe
Short ssDNA oligonucleotide complementary to target gene
56
Probe parts
- Reporter molecule (5'): Fluorophore - Quencher molecule (3'): Absorbs emitted fluorescence
57
Sequence specific approach when Probe is intact
1) Reporter excited by λ1 emitting λ2 2) λ2 absorbed by Quencher 3) No signal is detected
58
Sequence specific approach when Probe is Degraded
1) During PCR, DNA polymerase with 5' exonuclease activity cuts probe 2) Reporter, Nucleotides & Quencher released 3) Fluorescence can be detected
59
Advantages of Sequence specific approach
- Always reliable - If probe not digested then no signal
60
Disadvantages of Sequence specific approach
A difference Probe is needed for each gene we want to analyze
61
Threshold intensity
Amount of PCR product that can be reliably detected during efficient phase
62
Threshold cycle
Cycle number at which the amount of PCR product reaches the threshold intensity
63
Types of Quantification
- Relative Quantification - Absolute Quantification (uses standard calibration curve)
64
DNA Microarray/Chip
Solid surface (glass/silicone) where thousands of known DNA sequences (probes) representing genes are immobilized in an organized grid/array
65
DNA Microarray method
1) RNA extracted from sample 2) Reverse Transcription to cDNA 3) cDNA labelled with fluorescent dye 4) Placed on microarray 5) If cDNA binds probes it means the gene was expressed (analysis of genome wide expression)

MCB (4 decks)

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