Exam 4: Lecture 2

Question 1

Terminating Translation

Answer 1

• ribosome must discriminate between sense codons and stop codons
• stop codons not recognized by tRNA only recognized by release factors
• at every codon ribosome is inserting tRNAs and release factors
• stop codon lined up within A pocket, no tRNA properly base pairs and all tRNAs expelled
• when release factor brought into A pocket, recognizes stop codon and stimulates release of protein from ribosome
• codon in A pocket not stop codon, release factors expelled and ribosome continues
• different release factors recognizes stop codons in prokaryotes
• all stop codons recognized by single release factor in eukaryotes

Question 2

Ribosome Recycling

Answer 2

• disassociation must happen to ribosome after polypeptide/protein is released from ribosome
• required for ribosome reuse

Question 3

RRF

Answer 3

• release of protein chain stimulates insertion of ribosome recycling factor (RRF) into A pocket (which still contains stop codon)
• RRF bound to elongation faction G which stimulates release of two tRNAs in E and P pockets
• RRF and EF-G displaced from ribosome
• mRNA releases and two halves of ribosome disassociate

Question 4

Why ribosomal disassociation?

Answer 4

• allows for free small ribosomal subunit to be bound by IF1 or IF3 (pro) or elF1 or ElF1A (euk)
• block E and A pockets and are important in building pre-initiation ribosome complex

Question 5

Antibiotics

Answer 5

• naturally occurring used to combat bacterial infections
• many target components of ribosomal and translation machinery
• some specifically target prokaryotic cells (bacterial) while others inhibit translation in both pro and euk. (side effects)

Question 6

Puromycin

Answer 6

• can function similar to tRNA and can insert into A side of ribosome
• can serve as target for formation of peptide bond
• growing polypeptide chain added to the antibiotic
• prevents additional amino acids from being attached to protein
• causes disassociation of truncated protein (usually non-functional) from ribosome
• overall translation levels reduced, cell dies

Question 7

Exon Junction Complex

Answer 7

• set of proteins that are deposited on mRNA transcript ~20 nucleotides upstream of each exon-exon junction during splicing
• don’t recognize specific sequence, but interact with splicing factors at exon-intron junctions
• introns spliced out, EJC proteins deposited near remaining exon-exon junction which serve as guideposts for advancing ribosome
• contact by ribosome with EJC signals there are more exonic sequences to translate

Question 8

Nonstop Mediated Decay

Answer 8

• no stop codon so ribosome continues to translate whole transcript including 3’ UTR and Poly A tail
• many lysines added which is interpreted as a signal to degrade both protein and mRNA template

Question 9

Nonsense Mediated Decay

Answer 9

• change of a sense codon into a stop codon (nonsense mutation)
• ribosome figures out if stop codon is correct or nonsense mutation
• achieved through interactions with EJC
• ribosome reaches stop codon, determines if EJC proteins are ahead, if no ribosome terminates translation normally, if yes mutant mRNA degraded by RNA specific exonnucleases

Question 10

Nonsense-suppressor tRNAs

Answer 10

• arise when mutations within tRNA genes change anticodon sequence
• study example in notes
• nonsense suppressor mutations results in fully functional protein and wild type animal in forced experiments in lab

Question 11

Nonsense Supression

Answer 11

• suppress nonsense stop codons by mutation sin eukaryotic release
• release factors do not recognize stop codons and in rare instances ribosome will accept insertion and pairing of amino acid bearing tRNA with stop codon
• means in rare instances mutation within release factor can result in rescue of a loss of function mutant

Question 12

Drosophila Example

Answer 12

• ventral side of embryo patterned with alternating zones of naked cuticle and denticle belts
• formation is result of careful gene expression patterns and reflect segmentation of animal into anterior and posterior segments
• wingless (wg) gen critical in process
• if you remove wg you lose all naked cuticle segments and have embryo half the size only with denticle belts
• overexpose wg along ventral surface, eliminate all denticle belts and left with only naked cuticle

Question 13

Nonsense Suppression in Drosophila

Answer 13

• ovexpress wg containing nonsense mutation has no effet on denticle patterns because truncated Wg protein is non-functional
• overexpose mutant wg gene in fly that’s mutant for either eukaryotic release factor, see partial elimination of dentile belts because nonsense mutation has been suppressed and full length Wg protein has been generated