2.3 Expression of Genetic Information

Question 1

RNA Interference (RNAi)

Answer 1

• Double stranded RNA sequences capable of destruction of select mRNAs
• Recognize mRNA with complementary sequence
• May be a primitive immune system
• Double stranded RNA cleaved into small interfering •RNAs (siRNA) by Dicer enzyme
• Antisense strand incorporated into protein complex (RISC)

Question 2

RNA Interference (siRNA) steps

Answer 2

Step 1: Double stranded RNA cleaved by endonuclease Dicer to form siRNA which has overhanging ends (Step 2)
Step 3: siRNA associates with RISC complex with helicase that unwinds double stranded RNA
Step 3: siRNA associates with RISC complex with helicase that unwinds double stranded RNA
Step 4: siRNA binds to complementary mRNA
Step 5: mRNA is cleaved

RISC = RNA Interfering Silencing Complex

Question 3

RNAi Therapy

Answer 3

Degradation of viral RNAs
Example: HIV

Degradation of mRNAs from mutated genes known to cause cancer
Example: Leukemia

Degradation of mRNAs for malfunctioning proteins
Example: Huntingtons

Question 4

Micro RNAs (miRNA)

Answer 4

• First discovered in C. elegans nematode
• Small RNA Sequence complementary to 3’ UTR of specific mRNA
• Sequence is highly conserved
• Synthesized at specific times during development
• May turn genes on and off
• Translational inhibitors
• miRNA is partially complementary to 3’ UTR of mRNA target
• Estimated that humans encode as many as 1000 distinct miRNAs

Question 5

RNA Interference (miRNA) Steps

Answer 5

Step 1:s.sRNA has complementary sequences so can fold back on itself to form d.sRNA with a stem loop
Step 2: psedo-d.sRNA is clever near terminal loop by drosha so it has 3’ overhang then transported out of nucleus into cytoplasm to be cleaved by DICER (overhang at both endsO
Step 3: siRNA associates with argonaut portion and pre-RISC complex
Step 4: separation of d.s.RNA strands and removal of the passenger strand called miRNA. miRNA binds to complementary region on mRNA target
Step 5: miRNA inhibits translation of message or the mRNA is degraded

miRNA are formed from RNA that has ability to fold back on itself
Associate with RISC complex and bind to mRNAs blocking translation
miRNAs are not precisely complementary like siRNAs

Question 6

miRNAs involved in many processes

Answer 6

• Developmental programming
• Patterning of nervous system
• Control of cell proliferation and cell death
• Cell differentiation

Question 7

siRNAs vs miRNAs

Answer 7

• siRNAs derived from double stranded product of virus or synthetic dsRNA
• miRNAs encoded by genomic region
• miRNAs target specific •mRNA transcripts
• miRNAs primary role is to regulate gene expression

Question 8

Piwi interacting (pi) RNAs

Answer 8

• Expressed in germ cells
• Suppress movement of transposable elements
• Associate with PIWI proteins
• Required for successful gamete formation
• Deletion of PIWI proteins leads to failure in gamete formation

Question 9

CRISPER

Answer 9

• Clustered Regularly Interspaced Short Palindromic Repeats
• Found in bacteria
• Remnants of bacteriophage genomes
• CRISPR RNA transcript cut into smaller guide sequences
• Associate with cas9 protein
• Target and cleave DNA with homology to guide RNA (primitive immune system)
• Can use to edit genes

Question 10

In vitro translation experiments uncovered code (Nirenberg)

Answer 10

•Used synthesized RNA transcripts and cell free system
•Example: poly U encodes poly-phi
•using polynucleotide phosphorylase and uracil, crate an artificial mRNA of pure uracil (UUU)
•add mRNA made of pure uracil to 20 different test tubes. Each tube also contains general components supporting protein synthesis
•

Question 11

Khorana followed up on Nirenberg experiments

Answer 11

• Synthesized mRNA with alternating bases
GUGUGUGUGUGU
•Possible codons are GUG and UGU Should encode a protein of two alternating amino acids.
•Alternating Cys and Val amino acids

Question 12

Nirenberg and Philip Leder developed a technique using ribosome-bound transfer RNAs (tRNAs)

Answer 12

• Synthesized short 3 nucleotide mRNAs
• Incubated with ribosomes and labeled charged tRNAs
• Identified which tRNA/amino acid bound to synthetic mRNA

Question 13

Stop Codon, Start Codons

Answer 13

Stop codons, don’t code for an a.a (UAA, UAG, UGA)

Start Codon: Methionine AUG

Question 14

Mutation Types

Answer 14

• Synonomous change = the code changes but it still makes the same a.a.
• Nonsononomous change = you change the code and it makes a new a.a.
• If you change into a stop codon it’s a nonsense mutation (no a.a. is made)
• Frameshift mutation = single base insertion or deletion, changes the others a.a. as well, throws off the reading frame