RNA Structure and Techniques Flashcards
1
Q
tRNAs
A
- Deliver amino acids to ribosome
- Each amino acid has at least one unique tRNA
- small polypeptide chain: 73-94 residuals each
- Contains modified bases and multiple interactions to create L shaped molecule
2
Q
Secondary structure of tRNA
A
- Forms cloverleaf shape
- Consist of acceptor stem, TpsiC loop, variable loop, anticodon loop, and D loop
3
Q
Acceptor stem
A
- Holds 5’ and 3’ end of tRNA molecule
- Three nucleotides CCA enzymatically attached to 3’ end of acceptor stem
- CCA sequence serves site for amino acid attachment
4
Q
TpsiC loop
A
- Contains conserved thymine, pseudouridine,
5
Q
Variable loop
A
- lacks a conserved sequence and exhibits varying length among different tRNA species
6
Q
Anticodon loop
A
- Anticodon responsible with base pairing to respective codon in mRNA
7
Q
Triple base pairing
A
- Occurs when three nucleotides interact with each other through H-bonding
- Upon tertiary folding, a third nucleotide can enter through major groove and interact with one of original bases
8
Q
Divalent metal ions
A
- Direct interaction: Positively charged metal cation attracted to negatively charged phosphate backbone of RNA. Causes local neutralization of backbone and increases nucleotide stability
- Indirect interaction: Divalent cations attract water molecules through electrostatic interactions -> engage in H-bond with RNA bases to increase stability
9
Q
Polyamines
A
- Polyamines have multiple amino groups -> protonated at physiological pH. Neutralize negatively charged backbones through electrostatic interactions thus stabilizing RNA structure
10
Q
rRNA
A
- Ribosomes are 2/3 RNA, 1/3 protein
- provide structural foundation for ribosomal proteins
- contain certain modified nucleotides including ribothymidine and pseudouridine
- Different species of rRNA referred to according to sedimentation coefficients
11
Q
Ribozymes
A
- Catalytically active RNA molecules that can independently cleave RNA backbone through transesterification reactions
12
Q
Riboswitches
A
- Allow mRNA molecule to regulate own transcriptional and translational processes
- During transcription, ligand bind to aptamer domain -> triggers formation of terminator stem loop -> acts as signal to stop transcription
- During mRNA translation, ligand bind to aptamer domain and form stem-loop structure. Structure blocks ribosome from binding to ribosome binding site -> inhibits translation
13
Q
Restriction Enzymes
A
Two classes:
- Endonucleases: cleave phosphodiester bonds within sequence, exonucleases remove nucleotides from ends of DNA molecules
- RE nomenclature: 1st letter is genus, 2nd and 3rd letter is species, 4th letter is strain, Roman numeral is order of discovery
14
Q
Types of Restriction Enzymes
A
- Type 1 cleaves randomly while type 2 and 3 restriction enzymes cleave DNA chains at selected sites
- Type 2 REs have no ATP requirement
- Cleavage can leave staggered or “sticky” ends or can produce “blunt” ends
- Restriction nucleases that recognize same site: isoschizomers
15
Q
Disease Diagnostics with Gel Electrophoresis
A
- Restriction analysis can be used to detect single base pair mutations
- Restriction cleavage sites may appear/disappear
- Can be used to detect carriers of mutated genes
16
Q
Solid Phase Synthesis
A
- Used to make synthetic DNA probes/primers
- Nucleotides modified to contain: di-isopropyl group(becomes good leaving group), BCE group to safeguard phosphate backbone during synthetic process, DMT group bound to 5’ end of incoming nucleotide, base protection for preservation of functional groups
17
Q
Steps of solid phase synthesis
A
- Monomer activation and coupling to protonate di-isopropyl group -> forms unstable phosphite triester intermediate
- Oxidation by I2: intermediate oxidized by I2 -> nucleotide successfully added to resin bound DNA chain
- Deprotection: 5’ DMT removal -> creates free 5’ OH group
- BCE and base protection also removed in final product
- Occurs in 3’ - 5’ direction
18
Q
Southern Blotting
A
- Visualization technique to analyze specific DNA sequences in sample
- Shows similarities between two samples
Steps:- DNA cleave by restriction enzymes
- Fragments separated by gel electrophoresis
- Transfer to nitrocellulose paper -> denatures DNA to single strand
- Incubate with radiolabelled probe
- Visualize with autoradiogram
19
Q
Fluorescent in situ hybridization
A
- Used to identify presence/absence of genes in genome
- Fluorescently tagged probes are hybridized to chromosomes and visualized using fluorescence microscope
- Used diagnostically to identify multiple copies of HER2 gene
20
Q
Maxam Gilbert Sequencing
A
- Radiolabel DNA sample for identification
- React sample with different reagents
- Dimethyl sulfate(neutral) cleaves G
- Dimethyl sulfate(acidic) cleaves both A and G
- Hydrazine + 1.5M NaCl cleaves C
- Hydrazine cleaves T
- Run samples on gel to identify order
21
Q
Sanger Sequencing
A
- Determines number of nucleotides in DNA
- Fragments created by synthesis of complementary strand
Steps:
- Radiolabeling
- Dideoxynucleotide treatment
- Electrophoresis and visualization
22
Q
Automated sequencing
A
- Similar to Sanger but more efficient
- uses non-radiolabeled primers, DNA polymerase, regular dNTPs, all four ddNTPs at low concentration
23
Q
Single Molecule Sequencing
A
- Uses fluorescently labeled dNTPs to determine sequence of DNA as it is synthesized
- Flow in one nucleotide at a time
24
Q
Electronic Sequencing
A
- Measures DNA synthesis by products
- When nucleotide is added, releases pyrophosphate and proton
- Released proton detected by ion sensor
25
Pyrosequencing
- Measures pyrophosphate released upon nucleotide incorporation
26
PCR
- DS DNA, Taq polymerase, MgCl, dNTPs, primers
- Separate DS DNA using heat -> DNA polymerase + annealing + primers -> 2 copies
27
qPCR
- Can both detect and quantify presence of certain genes in real time
- Measurements made during each cycle of polymerization
- Exponential increase in product can quantify number of copies
28
Cloning
- Insert gene of interest into suitable plasmid by treating both with same restriction enzyme
- Deliver recombinant DNA molecules into bacteria
- Check for cells that have taken up vector by antibiotic resistance
- Plasmids are then replicated, or cloned by DNA synthesizing machinery of the host
- Fusion proteins can be expressed and purified
