Splicing and Translation 1 Flashcards
1
Q
Splicing Mutations
A
- Mutations that affect splicing responsible for 15% of genetic diseases
- Thalassemia caused by splicing mutation in hemoglobin subunit -> reduction of RBCs -> anemia
- Thalassemia causes A-> G mutation in intron of beta-globulin subunit -> creates new 5’ splice site
- 50% U1 assembles on original and 50% U1 assemble on new
- If original splice site recognize -> normal beta globulin translated
- If mutated recognized, premature STOP codon in transcript -> truncated non functional beta globulin
2
Q
Tissue specific splicing
A
- Calcitonin is hormone that regulates calcium and phosphorus metabolism
- Calcitonin gene related peptide regulates vasodilation in brain
- Made from same primary transcript -> undergo tissue specific splicing and alternative polyadenylation -> contain two poly(A) sites
- In thyroid gland: first poly(A) site recognized, includes exons 1, 2, 3, 4
- In brain: second poly(A) site recognized producing mRNA that includes exons 5 and 6
3
Q
Evolution of splicing
A
- Spliceosome evolved from self-splicing introns, allows for more intricate regulation of splicing -> influenced by snRNPs and CTD
- Higher order eukaryote use spliceosome, lower use self splicing
- Do not require proteins and considered ribozymes
- Classified into Type 1 and Type 2
4
Q
3D structure of self splicing RNA
A
- Structure depends on double helices with stems and loops
- Heating RNA: When 3D structure is denatured, lose catalytic function
- Using denaturing agents: Chemical agents that disrupt base pairing result loss in catalytic activity
- Altering essential nucleotides reduces catalytic activity
5
Q
Group 1 self splicing introns
A
- Occurs using two transesterification reactions using a guanosine cofactor
- Formation of catalytic site: Internal guide sequence (IGS) within intron base pairs with 3’ region of upstream exon adjacent to 5’ splice site. Folding forms catalytic site for guanosine cofactor binding
- First transesterification reaction: G cofactor enters active site and aligned so 3’OH attacks phosphate group of 5’ splice site. Induces cleavage of intron and incorporation of guanosine cofactor at 5’ end
- Second transesterification reaction: 3’ OH of upstream exon attacks phosphate group of 3’ splice site to release linear intron and join two exons. Does not form lariats
6
Q
Group II self splicing introns
A
- 2’OH group of adenine nucleotide performs nucleophilic attack at 5’ splice site to form lariat structure
- 3’OH of exon 1 attacks 5’ of exon 2 to release lariat and join exons
- Similar to spliceosome splicing
7
Q
Comparison of splicing mechanisms
A
- All: TE1: ribose OH attacks 5’ splice site. TE2: 3’OH of exon 1 attacks 3’ splice site on intron to join exon 1 and 2
- Attacking unit: 3’OH of guanosine cofactor for Type 1. 2’OH of key A residue for Type 2 and spliceosome
- Type 1 does not form lariat, while Type 2 and spliceosome does
- Type 1 and Type 2 does not use proteins. Spliceosome uses proteins
- Type 1 has L19 catalytic byproduct in tetrahymena rRNA. No catalytic byproduct for Type 2 and spliceosome
8
Q
Catalytic activity of group 1 self spliced introns
A
- In tetrahymena, group 1 self splicing introns initially released as linear RNA molecule containing 414 nucleotides
- First splicing event: 414 nucleotide RNA loses 15 nucleotides
- Second splicing event: Loses additional 4 nucleotides
- Total loss of 19 accounts for designation “L19” for final molecule
9
Q
L19 can undergo nucleotidyl transfer reactions
A
- Lenghten RNA molecule by stealing nucleotides from different substrate
- Base pairing with first substrate: 5 cytosine base RNA substrate binds with G rich 5’ end of L19 RNA. Substrate reffered to as C5
- Shortening of first substrate: 3’OH of L19 nucleophilically attacks C5 at terminal cytosine. Transesterification cleave bond between terminal C and rest of substrate and form bond between terminal C and 3’ end of L19. C5 shortened to 4 cytosines and released
- Base pairing with second substrate: New C5 substrate binds with L19
- Lengthening of second substrate: Second transesterification where 3’OH of new C5 attacks 3’ end of L19. Cleaves bond between C and L19, and form bond between C and 3’ end of new C5. C5 lengthened to 6 cytosines and L19 back to normal
10
Q
Tetrahymena rRNA splicing is magnesium dependent and protein independent experiment
A
- Background: 26S pre-rRNA contains intervening sequence (IVS). Determine if process magnesium dependent or on proteins
- Methods: Negative control has unspliced pre-rRNA transcript, positive control has linear and circular form of spliced out IVS
- Results: IVS products only generated in presence of magnesium ions even with presence of proteins
11
Q
Guanosine cofactor forms a triple base pair in active site experiment
A
- Background: In type 1, guanosine cofactor must bind to active site(P7) to facilitate first transesterification reaction. Active site formed from G:C Watson base pairing
- Methods: RNA constructs (wild type and two mutants) tested for activity using guanosine cofactor or 2-aminopurine cofactor
- Results: Wild type had most efficient splicing in presence of guanosine. Single mutant had only one mutation and nucleotides were A:C. No WC base pairing meant no catalytic activity. Double mutant restored instrastrand WC base pairing, nucleotides were A:U. Minimal catalytic activity in presence of guanosine cofactor. Catalytic activity restored with 2AP cofactor -> resulted in same valence electrons in same configuration as WT RNA+ G cofactor
12
Q
Magnesium is essential for self splicing catalysis
A
- Methods: bond formed between 3’ oxygen of guanosine cofactor and phosphorus of 5’ splice site. bond broken between phosphorus and 3’ oxygen of upstream RNA. Oxygen carries negative charge, needs to be stabilized by divalent cation. 3’ oxygen substituted with sulfur, if Mg2+ needed, no catalytic activity
- Results: Sulfur and normal RNA exposed to Mg2+ and guanosine cofactor. Self splicing occurred with normal RNA, but none with sulfur. Positive control using manganese, can coordinate to both atoms -> expected splice products for both.
- Conclusion: Mg2+ essential at active site for group 1 splicing
13
Q
Mechanism of polypeptide elongation
A
- Eukaryotic consist of 40S small subunit for mRNA interaction and 60S large subunit to catalyze formation of peptide bond between amino acids
- A(amino-acyl) site: where charged tRNAs enter ribosome
- P(peptide) site: where peptide bonds are formed
- E(exit) site: where uncharged tRNA leave ribosome
- Charged tRNA also called amino-acyl tRNAs, amino group bound via acyl linkage
- amino group of AA at A site and carbonyl group of AA at P site undergo dehydration synthesis reaction, forming amide linkage and releasing water
- Extends polypeptide chain from N terminal to C terminal
- After linkage, tRNA shift one position forward. A -> P, P -> E. Allows for synthesis in 5’-3’ direction
- Accuracy of synthesis dependent on formula p=(1-e)^n. e is error rate, and n is amino acids
- Error rate must not exceed 10^-4
14
Q
Elucidating genetic code
A
- Holley, Khorana, and Nirenberg made series of polypeptides made of trinucleotide repeats
- Depending on order of nucleotides in repeat, amino acid comprising polypeptide would be different
15
Q
Reading genetic code
A
- Three nucleotides is ideal length for 20 amino acids
- If 2 nucleotide long, only 16 possibilites
- If 4 long, 256 possibilites
- 3 gives 64 possibilities, with three that serve as STOP signals
- Read in 5’-3’ direction
- Pair in complementary and antiparallel manner, first base of codon binds to third base of anticodon
16
Q
Key features of genetic code
A
- Unambiguous: three nucleotide codon only encodes one amino acid. Each tRNA only carries one specific amino acid
- Degenerate: Many unique codons can specify same amino acid. Differ in nucleotide at third position -> helps minimize the effects of mutations. Tryptophan and Methionine exceptions because only encoded by one codon. Codons similar in sequence code chemically similar amino acids
- Non-overlapping: Codons read in sequential manner without overlapping. Helps prevent effect of mutations
17
Q
Charging tRNA - Class I vs Class II
A
- 20 different amino-acyl tRNA synthetase enzymes attach correct amino acid to tRNA
- Divided into Class I and Class II, uses ATP to catalyze different reaction to create acyl linkage between amino acid and corresponding tRNA
- 10 AARS belong to Class I and other 10 to Class II
- Activation of amino acid: Both classes activate amino acid substrate via adenylation. ATP -> AMP and pyrophosphate, hydrolyzation carries reaction forward. AMP is attached to amino acid at carbonyl group. Activated amino acid covalently linked to synthetase through R group -> formation of enzyme bound aminoacyl adenylate
- Charging tRNA molecule: After amino acid activated, two class of AARS charge tRNA at CCA arm
- Class I: 2’OH of adenine nucleotide in CCA acceptor nucleophilically attacks carbonyl group of amino acid releasing AMP. Transesterification moves amino acid onto 3’OH of adenine nucleotide. Monomeric
- Class II: 3’OH of A nucleotide of CCA attacks carbonyl group, no transesterification reaction. Dimeric
- Nomenclature: tRNA that specificies Alanine is tRNAAla, if incorrectly charged with serine -> serinyl-tRNAAla
18
Q
Thr-tRNA synthetase
A
- Need to distinguish from similar AA (valine and serine)
- Zinc ion: active site contains zinc ion coordinated to enzymes two histidine residue and cysteine residue. zinc ion coordinated to Thr substrate via amino and R hydroxyl group. Serine can coordinate but valine cannot
- Key aspartic residue: Aspartic residue in active site hydrogen bonds with R-hydroxyl group of amino acid substrate. Serine can be mischarged since it mimics interactions.
- Proofreading site: Editing site 20A away from active site. Catalyzes hydrolysis of aminoacyl linkage between Thr tRNA and incorrectly charged amino acid. Serinyl-tRNA undergo hydrolysis to release serine and tRNAthr. Only charged molecules smaller than tRNA can enter.
19
Q
Ile-tRNA synthetase
A
- Similar size discrimination mechanism of Thr-tRNA synthetase
- Amino acids larger than Ile rejected from active site
- Smaller molecules go to editing site
- Ile-tRNA synthetase performs similar editing as exonuclease domain in DNA polymerase by removing nucleotide by flipping strand
20
Q
Ala-tRNA synthetase
A
- Recognizes tRNAAla through mechanism that doesn’t involve anticodon region
- Relies on micro-helix in acceptor stem, specifically G3:U70 base pair.
- If G3:C70 in tRNACys changed to G3:U70, mutated tRNACys mischarged with alanine
