Transcript Exam II Review: - Texas Tech University

Covers :
1.
RNA Processing
2.
Translation
3.
Genetic Engineering
4.
Membrane Transport
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.
1. Purposea. Protect mRNA from nucleolytic degradation in the
cytosol.
b. Aid the ribosome in selecting translational start site.
1. RNA Triphosphatase
2. Capping Enzyme
3. Guanine-7-Methyltransferase
4. S-Adenosylmethionine (SAM)
5. 2’-O-Methyltransferase
1.
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.
1. 5’ cap is added shortly after initiation of RNA synthesis
in the nucleus.
Purpose1. To protect mRNA from nucleolytic degradation in
the cytosol.
2. Marks mRNA for nuclear export.
3. Aids in ribosomal recognition.
1. Cleavage and Polyadenylation Specifity Factor (CPSF)
2. Poly (A) Polymerase (PAP)
3. Poly (A) Binding Protein (PABP)
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.
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.
Purpose1. Pre-mRNA has noncoding sequences that must be
cut out from Eukaryotic mRNA before it can be
read by the ribosome.
1. Spliceosome Complex2. Small Nuclear RNAs (snRNAs)
3. Small Nuclear Ribonuclear Proteins (snRNPs/Snurps)
4. U1
5. U2
6. U3
7. U4
8. U5
9. U6
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.
Purpose1. Ribosomes orchestrate translation of mRNA to
synthesize proteins.
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
Purpose1. 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
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.
1. Secondary- 4 domain flower
2. Tertiary-Numerous lobes, channels and tunnels
a. A site- Accommodates incoming aminoacyltRNAs
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
Purpose1. 3 base anticodon determines mRNA and amino
acid binding.
2. When charged, amino acids bind to tRNA by
ester bonds
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 codonanticodon 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.
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
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.
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.
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.
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.
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.