Covers :1.RNA Processing2.Translation3.Genetic Engineering4.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. Purpose- a. Protect mRNA from nucleolytic degradation in

the cytosol.b. Aid the ribosome in selecting translational start

site.

1. RNA Triphosphatase2. Capping Enzyme3. 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.

Purpose-

1. 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 polymerase3. PABPs associate with Poly (A) tails in the cytosol to

organize them into nucleoprotein particles.

Purpose- 1. Pre-mRNA has noncoding sequences that

must be cut out from Eukaryotic mRNA before it can be read by the ribosome.

1. Spliceosome Complex- 2. Small Nuclear RNAs (snRNAs)3. Small Nuclear Ribonuclear Proteins

(snRNPs/Snurps) 4. U15. U26. U37. U48. U59. 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.

Purpose-1. Ribosomes orchestrate translation of

mRNA to synthesize proteins.

1. Ribosome2. tRNA3. Aminoacyl-tRNA Synthase4. IF-15. IF-26. IF-37. EF-Tu8. EF-Ts9. EF-G10. RF-111. RF-212. RF-313. RRF14. Ubiquitin15. Proteosome16. HSP 7017. HSP 6018. Chaperone Proteins

Purpose- 1. Bind mRNAs such that its codons can be

read with high fidelity. 2. Has specific binding sites for tRNA

molecules3. Mediation of interactions of nonribosomal

protein factors that promote initiation, elongation and termination of polypeptide.

4. Catalyze peptide bond formation5. Moves to translate sequential codons

1. Prokaryotica. Small subunit (30S)- 16S rRNA + 21 proteinsb. Large subunit (50S)- 5S and 23S rRNA + 31

proteins-Proteins rich in K & R amino acid residues

2. Eukaryotica. Small subunit (40S)- 18S rRNA + 33 proteinsb. Large subunit (60S)- 28,5.8 and 5S rRNAs + 49

proteins-More complex because euk. Translation is more

complex.

1. Secondary- 4 domain flower2. Tertiary-Numerous lobes, channels and

tunnelsa. A site- Accommodates incoming aminoacyl-tRNAs b. P site- Accommodates incoming peptidyl-tRNAsc. E site- Accommodates deacylated tRNAs

3. Small subunit- -Purpose: Binding tRNAs and ribosomal recognition

4. Large subunit-Purpose: Mediates chain elongation

Purpose-1. 3 base anticodon determines mRNA and

amino acid binding.2. When charged, amino acids bind to tRNA

by ester bonds

1. Secondary- Cloverleafa. 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. Tertiarya. 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 acid1. 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. Initiationa. 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. Elongationa. Elongation factors bind all tRNAs except

start codons. b. Requires GTPc. 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. Terminationa. 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.