Exam II Review:

1
Exam II Review

• Covers
• RNA Processing
• Translation
• Genetic Engineering
• Membrane Transport

2
RNA Processing

• 1. Purpose
• a. mRNA in the nucleus is not Translationally
  Competent. The primary transcript (or pre-mRNA)
  must go through (5 Capping, Polyadenylation and
  intron splicing) in order to be ready for the
  ribosome in the cytosol.

3
5 Capping

• 1. Purpose-
• a. Protect mRNA from nucleolytic degradation in
  the cytosol.
• b. Aid the ribosome in selecting translational
  start site.

4
Proteins involved in 5 Capping

• 1. RNA Triphosphatase
• 2. Capping Enzyme
• 3. Guanine-7-Methyltransferase
• 4. S-Adenosylmethionine (SAM)
• 5. 2-O-Methyltransferase

5
Mechanism of 5 Capping

• RNA Triphosphatase Removes leading phosphate
  group from mRNAs 5 terminal triphosphate group.
  
• 2. Capping Enzyme- Guanylates the mRNA, creating
  5-5 Triphosphate Bridge when it hydrolyzes GTP.
  
• 3. Guanine-7-Methyltransferase- Uses SAM to
  methylate guanine.
• 4. 2-O-Methyltransferase- Uses SAM to methylate
  the 1st and 2nd nucleotides of the pre-mRNA.

6
Additional Notes on 5 Capping

• 1. 5 cap is added shortly after initiation of
  RNA synthesis in the nucleus.

7
Polyadenylation (AAUAAA)

• Purpose-
• 1. To protect mRNA from nucleolytic degradation
  in the cytosol.
• 2. Marks mRNA for nuclear export.
• 3. Aids in ribosomal recognition.

8
Proteins Involved in Poly (A) Tail

• 1. Cleavage and Polyadenylation Specifity Factor
  (CPSF)
• 2. Poly (A) Polymerase (PAP)
• 3. Poly (A) Binding Protein (PABP)

9
Mechanism of Polyadenylation

• 1. CPSF- cleaves 15-25nt past AAUAAA and 50nt
  before U/GU sequences, which activates PAP.
• 2. PAP- Adds AAUAAA tail to 3 OH groups.

10
Additional Notes on Polyadenylation

• 1. Cleavage and Polyadenylation are coupled.
• 2. PAP is a template-independent RNA polymerase
• 3. PABPs associate with Poly (A) tails in the
  cytosol to organize them into nucleoprotein
  particles.

11
Intron Splicing

• Purpose-
• 1. Pre-mRNA has noncoding sequences that must be
  cut out from Eukaryotic mRNA before it can be
  read by the ribosome.

12
Proteins Involved in Intron Splicing

• 1. Spliceosome Complex-
• 2. Small Nuclear RNAs (snRNAs)
• 3. Small Nuclear Ribonuclear Proteins
  (snRNPs/Snurps)
• 4. U1
• 5. U2
• 6. U3
• 7. U4
• 8. U5
• 9. U6

13
Mechanism of Intron Splicing

• 1. Lariat Structure- U1 recognizes 5 end of
  intron, U2 recognizes branch point adenine. A 2,
  5 phosphodiester bond forms between introns
  adenosine residue, the exon is thereby released
  while the intron forms a lariat structure.
• 2. Splice Product- The 5 exons free 3 OH group
  displaces the 3 end of the intron, forming a
  phosphodiester bond with the 5 terminal
  phosphate of the 3 exon, yielding the spliced
  product. The intronic lariat is released with its
  3 OH group and is rapidly recycled.

14
Translation

• Purpose-
• 1. Ribosomes orchestrate translation of mRNA to
  synthesize proteins.

15
Proteins Involved in Translation

• 1. Ribosome
• 2. tRNA
• 3. Aminoacyl-tRNA Synthase
• 4. IF-1
• 5. IF-2
• 6. IF-3
• 7. EF-Tu
• 8. EF-Ts
• 9. EF-G
• 10. RF-1
• 11. RF-2
• 12. RF-3
• 13. RRF
• 14. Ubiquitin
• 15. Proteosome
• 16. HSP 70
• 17. HSP 60
• 18. Chaperone Proteins

16
Ribosomes

• Purpose-
• 1. Bind mRNAs such that its codons can be read
  with high fidelity.
• 2. Has specific binding sites for tRNA molecules
• 3. Mediation of interactions of nonribosomal
  protein factors that promote initiation,
  elongation and termination of polypeptide.
• 4. Catalyze peptide bond formation
• 5. Moves to translate sequential codons

17
Prokaryotic v. Eukaryotic Ribosomes

• 1. Prokaryotic
• a. Small subunit (30S)- 16S rRNA 21 proteins
• b. Large subunit (50S)- 5S and 23S rRNA 31
  proteins
• -Proteins rich in K R amino acid residues
• 2. Eukaryotic
• a. Small subunit (40S)- 18S rRNA 33 proteins
• b. Large subunit (60S)- 28,5.8 and 5S rRNAs 49
  proteins
• -More complex because euk. Translation is more
  complex.

18
General Ribosomal Structure

• 1. Secondary- 4 domain flower
• 2. Tertiary-Numerous lobes, channels and tunnels
• a. A site- Accommodates incoming aminoacyl-tRNAs
  
• b. P site- Accommodates incoming peptidyl-tRNAs
• c. E site- Accommodates deacylated tRNAs
• 3. Small subunit-
• -Purpose Binding tRNAs and ribosomal recognition
• 4. Large subunit
• -Purpose Mediates chain elongation

19
Transfer RNAs (tRNAs)

• Purpose-
• 1. 3 base anticodon determines mRNA and amino
  acid binding.
• 2. When charged, amino acids bind to tRNA by
  ester bonds

20
tRNA Structure

• 1. Secondary- Cloverleaf
• a. 5 terminal phosphate group.
• b. Acceptor Stem- Amino acid covalently attached
  to its 3 terminal OH group.
• c. D Arm- Dihydrouridine
• d. Anticodon Arm- Contains anticodon sequence, 3
  purine is invariably modified.
• e. T Arm- Psuedouridine
• f. CCA Sequence- 3 sequence with free OH group.
• g. 15 invariant/8 variant positions- Only
  purine/pyrimidine.
• h. Variable Arm- Base modifications help promote
  attachment of proper amino acid to the acceptor
  stem and strengthen codon-anticodon interactions.
  
• 2. Tertiary
• a. L shape in which acceptor Stem/T Arm stems
  from one leg and D Arm/Anticodon Arm stems from
  the other.
• b. Maintained by extensive stacking interactions
  and non-Watson-Crick associated base pairing
  between helical stems.

21
tRNA Function

• 1. Charged tRNAs carry amino acids to the
  ribosome
• Mechanism
• Aminoacyl-tRNA Synthetase- Produces the charged
  amino acid
• 1. AA ATP ? AA-AMP Pyrophosphate (2Pi)
• 2. AA-AMP ? AA-tRNA AMP

22
Additional Notes on Mechanism of Aminoacyl-tRNA
Synthetase

• 1. AA-tRNA (Aminoacyl-adenylate) is a high energy
  compound.
• 2. The overall reaction is driven to completion
  by the hydrolysis of 2Pi generated in step a.

23
Translation Mechanism (1)

• 1. Initiation
• a. Binding to start codon (AUG/Met)
• b. Small subunit finds Kozac sequence (ACCAUGG)
  (Shine-Dalgarnoprok. AGGAGG).
• Proteins
• IF-1 Assists IF-3.
• IF-2 Binds to initiator tRNA start codon
  (AUG/Met) and GTP.
• IF-3 Releases mRNA and tRNA from subunit.

24
Translation Mechanism (2)

• 2. Elongation
• a. Elongation factors bind all tRNAs except start
  codons.
• b. Requires GTP
• c. Peptide bonds catalyzed by peptidyl
  transferase activity of large subunit.
• d. Polypeptides synthesizes about 40AA/second.
• Proteins
• EF-Tu Binds AA-tRNA to GTP at A-site.
• EF-Ts Displaces GDP from EF-Tu.
• EF-G Promotes translocation through GTP binding
  and hydrolysis.

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Translation Mechanism (3)

• 3. Termination
• a. Release factors mimic tRNAs and bind to stop
  codons.
• b. Release factors use GTP to bind the protein to
  water, terminating the protein chain.
• Proteins
• RF-1 Recognizes UAA UAG stop codons.
• RF-2 Recognizes UAA UGA stop codons.
• RF-3 Stimulates RF- 1 2 release via GTP
  hydrolysis.
• RRF Together with EF-G, induces ribosomal
  dissociation of small and large subunits.

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Post-Translational Modification

• 1. Protein folding occurs as it is being
  synthesized.
• 2. Protein is facilitated by chaperone proteins
  that prevent interaction of protein with other
  molecules.
• a. HSP70 and HSP60 use ATP to bind and unbind
  folding protein.
• b. Protein folding errors cause diseases.
• c. Ubiquitin and proteosomes function to degrade
  proteins.
• 3. Translation can also be modified by
• a. Initiation factor repressors.
• b. Translational repressors.
• c. Regulation of mRNA half-life.
• d. Nonsense-mediated decay (NMD).
• Prevents translated of improperly processed
  mRNAs.