FINAL EXAM Flashcards
Goal of RNAi in Cells (2)
1) Defends against viruses, invading RNA, and transposable elements.
2) Represses/Silences gene expression through mRNA degradation or by translation inhibition.
Goal of RNAi in the Lab (1: A,B)
1) Knock-down gene expression
A) Study gene function
-Fxn of Ras
B) Therapeutic Treatment
-siRNA to treat cancer
-siRNA against VEGF ^glaucoma
How does RNAi silence gene expression?
By triggering chromatin modifications
When are transposable elements degraded by RNAi?
During transposition
When is mRNA destroyed by RNAi?
Before translation of that mRNA occurs.
miRNA
(micro RNA)
1) Where is it located…
2) Structure looks like what…
3) Targets what…
1) Encoded in genome
2) Short-hairpin RNA structure during processing
3) Targets a variety of mRNAs
siRNA
(short/small interfering RNA)
1) Where is it located/come from…
2) Used in/for what…
3) Targets what…
1) Processed from a longer exogenous dsRNA taken up by Cells
2) Used in knock-down gene expression
3) Targets only one or few mRNAs
piRNA
(piwi interacting RNA)
Function?
Selectively specific for transposable elements
epigenetic silencing
Which 2 RNAs discussed interact with RISC?
miRNA and siRNA
What is pre-miRNA?
The hairpin segment of a larger transcript
What are used in the RNAi Pathway?
(miRNAs (4) and processing parts (2;3)
pri-miRNA, pre-miRNA, duplex-miRNA, mature miRNA
Drosha, RISC (Dicer, Argonaute, Armitage)
What is RISC?
RNA-induced silencing complex
-made up of: Dicer, Argonaute, and Armitage
What is the function of Drosha?
Drosha processes pri-miRNA into smaller segments of pre-miRNA in the nucleus
What happens to pre-miRNA once it is exported from the nucleus?
Attaches to RISC in cytoplasm
What is the function of Dicer in RISC?
Dicer processes pre-miRNA into shorter duplex-miRNA.
What is and what is the function of Armitage?
Armitage is a helicase.
Armitage unwinds (denatures) the duplex miRNA to form mature miRNA
What is the function of Argonaute?
Argonaute cuts the mRNA and the mRNA is degraded
What occurs before the Argonaute can cut the mRNA?
The mature-miRNA complementary base pairs with the target mRNA
2 Fates of RNAi Pathway
1) mRNA degradation due to Argonaute cutting it. <–strong complementary base pairing
2) translation blocked <–mismatches in complementary base pairing
Which RNAi Pathway fate do siRNA and miRNA typically follow?
siRNA typically follows the path of mRNA degradation
miRNA typically follows the path in which translation is blocked
How many base pairs long is the miRNA after cleavage by both Drosha and Dicer?
22 base pairs in resulting hairpin structure
Dicer is compared to a hatchet.
What are the parts associated with it and their function(s)?
Blade
-(RNAse domains, cuts pre-miRNA)
Handle/”Ruler Helix”
-measures the length of the miRNA
(Positioning more than length)
PAZ domain 3’ end of RNA
-contains positively charged amino acids (Lys and Arg)
-Residues hold the 3’ end of miRNA
What occurs when RdRP is present during the RNAi Pathway?
RdRP (RNA dependent RNA polymerase) elongated miRNA/siRNA to form more dsRNA
instead of degradation following complementary base pairing; miRNA/siRNA is elongated and processed by Dicer once more to create new miRNAs/siRNAs
What are some ways to measure how much mRNA is in a sample? (5)
-qPCR
-Fluorescent in situ hybridization (FISH)
-Northern blotting
-RT-PCR
-RNA-seq (used to quantify/identify lots of transcripts)
What components is an 80S eukaryotic ribosome made up of?
The large subunit (60S) and the small subunit (40S); which use proteins.
In a eukaryotic ribosome, the large subunit (60S) contains what? (4)
- 5.8 S rRNA (160 nts)
- 5 S rNA (120nts)
- 28 S rRNA (4700 nts)
- 49 proteins
In a eukaryotic ribosome, the small subunit (40S) contains what? (2)
- 18S rRNA (1900 nts)
- ~33 proteins
What is special about the 18S rRNA in the small subunit (40S)?
The 18S rRNA increases hydrogen bonding interactions
What components are found in the prokaryotic ribosome (70S)?
The large subunit (50S) and the small subunit (30S)
In the prokaryotic ribosome, the large subunit (50S) contains what? (3)
- 5S rRNA (120 nts)
- 23S rRNA (2900 nts)
- ~34 proteins
In the prokaryotic ribosome, the small subunit (30S) contains what? (2)
- 16S rRNA (1540 nts)
- 21 proteins
What is special about the 16S rRNA found in a prokaryotic ribosome?
- Allows for reconstruction
- Sequenced to identify species in a mixed pool of organisms
What evidence supports that the ribosome is a ribozyme? (3)
- If proteins are removed, txn still occurs.
- No amino acids are present at the active site (which would play an active role in catalysis).
- The 28S rRNA is a peptidyl transferase (performs catalysis) and found in the large subunit (60S) of the eukaryotic ribosome.
What are the 5 overall stages of translation?
1) Activation of amino acids
2) Initiation
3) Elongation
4) Termination
5) Protein Folding
In translation, what occurs in the activation of amino acids stage?
The tRNA is aminoacylated
In translation, what occurs during initiation?
Both the mRNA and aminoacylated tRNA bind to the small ribosomal subunit followed by the binding of the large subunit.
In translation, what occurs during elongation?
Successive cycles of aminoacyl-tRNA binding and a peptide bond formation occur
In translation, what occurs during termination?
When a STOP codon is reached, translation stops. The mRNA and protein dissociate while the large and small subunits are recycled.
What are polysomes?
Multiple ribosomes that process/translate the same single strand of mRNA
In translation, where does the amino acid attach to on the tRNA?
The ccA 3’ end (amino acid arm)
In translation, what is responsible for attaching the tRNA to its cognate (matching) amino acid?
An aminoacyl tRNA synthetase
