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Exam II Review:

Exam II Review:. Covers : RNA Processing Translation Genetic Engineering Membrane Transport. RNA Processing. 1. Purpose:

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Exam II Review:

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  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-tRNASynthase 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-tRNASynthetase 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-Dalgarno=prok. 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 peptidyltransferaseactivity 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.

  25. 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.

  26. 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.

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