PTGR old exams

Question 1

1. three main differences between eukaryotic and prokaryotic mRNA

Answer 1

1. Eukaryotic mRNA has 3’ poly(A) tail and 5’ cap
2. Eukaryotic mRNA is spliced
3. Prokaryotic mRNA has Shine-Dalgarno sequence
4. Prokaryotic mRNA has multiple start and stop codons, eukaryotic only has one

Question 2

1. 3 eukaryotic and prokaryotic GTPase initation factors and their functions

Answer 2

1. Prokaryotic
   
   1. IF2:
      
      1. recognizes the fMet-initiator tRNA and complexes it with GTP
      2. stimulates association of 50S ribosomal subunit, leading to the hydrolysis of IF2 bound GTP
      3. this leads to the initiator tRNA being released by IF2 and accomadates into the P-site and initiation complex is formed
2. Eukaryotic
   
   1. eIF2
      
      1. GTPase, escorts Met-tRNA onto 40S subunit
   2. eIF5B
      
      1. GTPase, mediates assembly of 80S from 40S and 60S

Question 3

1. name 3 prokaryotic peptides that induce ribosome stalling

Answer 3

1. Note: ribosome becomes stalled due to absence of a codon in decoding center due to:
   
   1. Stop codon is missing
   2. Stop codon is read through
   3. Stalling on intact mRNA -> cleavage to form non-stop complex
2. oligo-proline
   
   1. is poor substrate as electron donor and acceptor during peptide bond formation
   2. ribosomes become stalled when synthesizing polypeptides containing multiple consecutive proline residues
3. TnaC
   
   1. Blocks binding of Rho by binding to UGA stop codon, inducing translational stalling
   2. Allows transcription of downstream genes TnaA/B
4. SecM
   
   1. Translational elongation stalling to induce translation initiation
   2. Acts as regulator for sec system
   3. If SecA is present, it acts as a force sensor preventing stalling, and preventing transcription of downstream genes (like those of SecA)
   4. If it is not present, then SecM binds and causes stalling, allowing transcription of downstream genes.

Question 4

1. 3 functions of mRNA localization

Answer 4

1. Localized translation
   
   1. Eg. Translation of β-actin at fibroblast leading edge
2. Asymmetric distribution of proteins to determine cell fate
   
   1. Eg. Distribution of Ash1 in yeast to determine mating type
3. Determination of embryonic axes (asymmetrical distribution of bicoid and oskar mRNA in drosophila)
   
   1. Developmental patterning
4. Avoiding unwanted translation
   
   1. Transport of myelin basic protein mRNA to processes of oligodendrocytes

Question 5

1. what process is the tmRNA involved in? describe briefly what other factors are necessary

Answer 5

1. Trans-translation: a ribosome rescue mechanism for non-stop complexes
   
   1. Ribosome resumes translation using tmRNA as a message
   2. Terminates at a stop codon at the end of the tmRNA reading frame and is recycled
   3. The mRNA and nascent polypeptide chain synthesized/used are targeted for destruction
2. tmRNA (transfer messenger RNA)
   
   1. functions similarly to both mRNAs and tRNAs
   2. Ef-Tu can bind to tRNA like domain
   3. Can tag peptides for degradation
3. Small protein B (SmpB)
   
   1. Associates with tmRNA to stabilize its structure
   2. Enhances affinity for alanyl-tRNA synthetase
   3. Is required for interaction with the non-stop complex genes encoding tmRNA (ssrA and smpB)

Question 6

1. what protein factors mediates the repressive action of the mRNA-bound miRNA-Ago complex (exact wording?) what other complexes are recruited and induce degradation of poly-A tail

Answer 6

Note: not sure if this is answer tehy want or not

1. protein factors
   
   1. RNA polymerase 2
      
      1. Transcribes pri-miRNA
   2. Drosha
      
      1. RNase III enzyme in combination with Pasha
      2. Cleavage of pri-miRNA to pre-miRNA
   3. Exportin5
      
      1. Export of pre-miRNA from the nucleus
   4. Dicer
      
      1. Along with dsRBD protein cleaves pre-miRNA into miRNA/miRNA duplex
      2. Dicer + dsRBD protein form RISC loading complex
   5. Hsp70/Hsp90
      
      1. Along with RISC, and with ATP hydrolysis, pass miRNA duplex to Ago protein
   6. One miRNA is cleaved from duplex, resulting in mature RISC complex
2. Degradation of poly-A tail
   
   1. PAN2/PAN3 complex
      
      1. Shortening till 100 As are left
   2. CCr4/Not Complex
      
      1. Until 30 As are left
   3. Exosome and Ski complex

Question 7

1. during alternative splice what factors lead to the inclusion of exons. Mechanism of proteins and how do they interact with each other/ splicing apparatus

Answer 7

1. splice factors are what regulate alternative splicing
   
   1. positive regulators (often SR proteins) lead to inclusion of exons
      
      1. SR proteins promote binding of U2 and U2AF
   2. negative regulators lead to exclusion (eg. hnRNPA1)
2. for example: sex determination in drosophila
   
   1. different Dsx proteins (produced via alternative splicing) are produced in males and females
   2. 3’ splice site of exon 4 has unusual recognition sequence, leading to it being skipped in males
   3. In females, that express Tra and Tra2, Tra2 binds to 3’ splice site, recruit further splicing factors

SR proteins promote binding of U2 and U2AF

Question 8

1. what is the mechanism of mRNA turnover in eukaryotes? with proteins mediating reaction

Answer 8

1. If mRNA translation is going on, it is protected via circularization, if not it is degraded
2. Regulated via exonucleases such as PAN2/PAN3, and Ccr4/NOT
   
   1. Remove poly(A) tail to allow degradation
3. the cytoplasmic exosome handles it
4. ARE (au rich element) binding proteins in the cytoplasm
5. Ski complex – channels RNA into the exosome
   
   1. Ski2: helicase that makes sure RNA unfolded to go into channel
   2. Ski3/Ski8 – protein interactions

Question 9

1. 3 pathways of mRNA surveillance in eukaryotes with what mistake in the mRNA induces them

Answer 9

1. Non-stop decay
   
   1. Missing stop codon -> Dom34/Hbs1/Ski7
2. Nonsense mediated decay
   
   1. Premature stop codon -> eRF1/eRF3
3. No go decay
   
   1. Stalling -> Dom34/Hbs1

Question 10

1. What criteria are used to predict microRNA targets? (IIIIII)

Answer 10

1. Complementarity with seed region
2. Conservation of seed region
3. Exclusion of highly structured mRNA regions

Question 11

1. explain “primary rRNA transcript” and name the two general processes by which rRNAs are processed (IIIII)

Answer 11

1. primary rRNA transcript is the first transcript formed by the RNA polymerase I while transcribing rRNA genes -> is several rRNAs in one transcript separated by short spacer sequences
   
   1. in eukaryotes: 18s rRNA, 5.8s rRNA, 28s rRNA
   2. in prokaryotes: 16s rRNA, tRNA, 23s rRNA
2. rRNA processing:
   
   1. rRNA precursor assembles with snoRNA enzymes which introduce modifications (such as methylations/pseudo uridylations) that aid in correct folding and assembly of the rRNAs
   2. cleavage of pre-rRNAs facilitated by snoRNAs to liberate individual rRNAs

Question 12

1. what is a MiRtron and how is it processed ? (IIII)

Answer 12

1. miRNA present in the intronic sequence of another gene
2. It is processed through splicing which bypasses drosha cleavage
3. Followed by debranching and cleavage by Dcr-1/Loqs to an miRNA-duplex
4. Cleavage of passenger strand by Ago-2 in the RISC complex gives rise to a mature ss miRNA.
5. short hairpin introns = mirtrons -> function like regular miRNA

Question 13

1. what is the size of the nuclear pore complex (in MDa), of how many proteins does it consist, and what is its symmetry (IIII)

Answer 13

1. size = 125 mDa
2. consists of 30-50 different proteins (also called nucleoporins)
3. 8 – fold symmetry

Question 14

1. Name three potential functions for mRNA localization (IIII)

Answer 14

1. developmental patterning through the asymmetrical distribution of cellular proteins
   
   1. e.g. embryonic axes definition in Drosophila through the asymmetrical distribution of oskar and bicoid
2. cell-fate decisions through the asymmetrical distribution of cellular proteins
   
   1. e.g. mating type selection in yeast through the asymmetrical distribution of Ash1
3. localized translation
   
   1. e.g. the localized translation of β-actin only at the leading edge of