Protein Synthesis - Biochemistry

Ribosome

Structure
- eukaryotes
  - small subunit (40S)
    - synthesized in the nucleus
  - large subunit (60S)
    - synthesized in the nucleolus
  - total = 80S
    - S = sedimentation
- prokaryotes
  - small subunit (30S) + large subunit (50S) → 70S
Catalytic sites
- A (acceptor) site
  - accepts charged aa-tRNA
- P (peptidyl) site
  - holds the growing peptide chain and transfers to the aa-tRNA in the A site
- E (exit) site
  - allows tRNA to exit the ribosome after releasing its amino acid to the aa-peptide chain
Forms
- polysomes
  - single mRNA being translated simultaneously by several ribosomes
  - can be
    - free
      - proteins for nucleus or mitochondria
    - membrane bound
      - found on rough ER
      - proteins for secretion or membrane insertion

Steps

Initiation
- initiation complex generated from
  - small ribosomal subunit
  - mRNA
    - 5'-cap (eukaryotes)
    - Shine-Dalgarno sequence (prokaryotes)
  - initiation aa-tRNA
    - met-tRNA (eukaryotes)
    - fmet-tRNA (prokaryotes)
  - initiation factors (IFs)
    - help in assembly
    - released when mRNA joins the ribosomal subunit
- once assembled, recruits the large ribosomal subunit
  - initiation aa-tRNA occupies the P-site
    - all other aa-tRNAs bind the A-site
  - the A-site is empty
Elongation
- aminoacyl-tRNA binds to A site
  - consumes 1 GTP
- peptidyl-bond formation
  - peptidyl transferase reaction catalyzed by ribosomal rRNA ("ribozyme")
  - transfers growing polypeptide to amino acid in the A site
- translocation
  - ribosome moves 3 nucleotides at a time in a 5' to 3' direction
  - moves peptidyl RNA from A site to P site
  - moves empty tRNA from P site to E site for exit
  - A site now empty and ready to accept next aa-tRNA
  - catalyzed by elongation factor-2 (eEF-2)
    - inactivated via ADP-ribosylation by bacterial toxins (Pseudomonas and Diphtheria)
  - consumes 1 GTP
- cycle is repeated for each amino acid addition to the polypeptide
- Wobble phenomenon
  - The anticodon of certain tRNA molecules can bind to multiple codons
  - The first two nucleotides of the codon adequately specify which tRNA (and amino acid) binds the codon, thereby permitting multiple nucleotides in the third position to bind the same tRNA.
Termination
- stop codon reached in mRNA
- release factor binds mRNA
- peptide bond is hydrolyzed and completed protein is released
- ribosomal complex dissociates
Overall energy requirement
- 4 high energy bonds
  - ATP to AMP for tRNA charging (2)
  - 2 GTP to GDP for elongation (2)

Pharmacologic Inhibition of Prokaryotic Protein Synthesis

Bind 30S
- aminoglycosides
  - inhibit formation of the initiation complex
  - promotes misreading of mRNA
Bind 50S
- chloramphenicol
  - inhibits peptidyl transferase
- macrolides
  - inhibits translocation
- clindamycin
  - inhibits translocation

References

Blood, Plasma, Serum	Reference Range
ALT	8-20 U/L
Amylase, serum	25-125 U/L
AST	8-20 U/L
Bilirubin, serum (adult) Total // Direct	0.1-1.0 mg/dL // 0.0-0.3 mg/dL
Calcium, serum (Ca²⁺)	8.4-10.2 mg/dL
Cholesterol, serum	Rec: < 200 mg/dL
Cortisol, serum	0800 h: 5-23 μg/dL //1600 h: 3-15 μg/dL 2000 h: ≤ 50% of 0800 h
Creatine kinase, serum	Male: 25-90 U/L Female: 10-70 U/L
Creatinine, serum	0.6-1.2 mg/dL
Electrolytes, serum
Sodium (Na⁺)	136-145 mEq/L
Chloride (Cl^-)	95-105 mEq/L
Potassium (K⁺)	3.5-5.0 mEq/L
Bicarbonate (HCO₃^-)	22-28 mEq/L
Magnesium (Mg²⁺)	1.5-2.0 mEq/L
Estriol, total, serum (in pregnancy)
24-28 wks // 32-36 wks	30-170 ng/mL // 60-280 ng/mL
28-32 wk // 36-40 wks	40-220 ng/mL // 80-350 ng/mL
Ferritin, serum	Male: 15-200 ng/mL Female: 12-150 ng/mL
Follicle-stimulating hormone, serum/plasma	Male: 4-25 mIU/mL Female: premenopause: 4-30 mIU/mL midcycle peak: 10-90 mIU/mL postmenopause: 40-250
pH	7.35-7.45
PCO₂	33-45 mmHg
PO₂	75-105 mmHg
Glucose, serum	Fasting: 70-110 mg/dL 2-h postprandial:<120 mg/dL
Growth hormone - arginine stimulation	Fasting: <5 ng/mL Provocative stimuli: > 7ng/mL
Immunoglobulins, serum
IgA	76-390 mg/dL
IgE	0-380 IU/mL
IgG	650-1500 mg/dL
IgM	40-345 mg/dL
Iron	50-170 μg/dL
Lactate dehydrogenase, serum	45-90 U/L
Luteinizing hormone, serum/plasma	Male: 6-23 mIU/mL Female: follicular phase: 5-30 mIU/mL midcycle: 75-150 mIU/mL postmenopause 30-200 mIU/mL
Osmolality, serum	275-295 mOsmol/kd H₂O
Parathyroid hormone, serume, N-terminal	230-630 pg/mL
Phosphatase (alkaline), serum (p-NPP at 30° C)	20-70 U/L
Phosphorus (inorganic), serum	3.0-4.5 mg/dL
Prolactin, serum (hPRL)	< 20 ng/mL
Proteins, serum

Hematologic	Reference Range
Bleeding time	2-7 minutes
Erythrocyte count	Male: 4.3-5.9 million/mm³ Female: 3.5-5.5 million mm³
Erythrocyte sedimentation rate (Westergren)	Male: 0-15 mm/h Female: 0-20 mm/h
Hematocrit	Male: 41%-53% Female: 36%-46%
Hemoglobin A1c	≤ 6 %
Hemoglobin, blood	Male: 13.5-17.5 g/dL Female: 12.0-16.0 g/dL
Hemoglobin, plasma	1-4 mg/dL
Leukocyte count and differential
Leukocyte count	4,500-11,000/mm³
Segmented neutrophils	54%-62%
Bands	3%-5%
Eosinophils	1%-3%
Basophils	0%-0.75%
Lymphocytes	25%-33%
Monocytes	3%-7%
Mean corpuscular hemoglobin	25.4-34.6 pg/cell
Mean corpuscular hemoglobin concentration	31%-36% Hb/cell
Mean corpuscular volume	80-100 μm³
Partial thromboplastin time (activated)	25-40 seconds
Platelet count	150,000-400,000/mm³
Prothrombin time	11-15 seconds
Reticulocyte count	0.5%-1.5% of red cells
Thrombin time	< 2 seconds deviation from control
Volume
Plasma	Male: 25-43 mL/kg Female: 28-45 mL/kg
Red cell	Male: 20-36 mL/kg Female: 19-31 mL/kg

Cerebrospinal Fluid	Reference Range
Cell count	0-5/mm³
Chloride	118-132 mEq/L
Gamma globulin	3%-12% total proteins
Glucose	40-70 mg/dL
Pressure	70-180 mm H₂O
Proteins, total	< 40 mg/dL

Sweat	Reference Range
Chloride	0-35 mmol/L
Urine
Calcium	100-300 mg/24 h
Chloride	Varies with intake
Creatinine clearance	Male: 97-137 mL/min Female: 88-128 mL/min
Estriol, total (in pregnancy)
30 wks	6-18 mg/24 h
35 wks	9-28 mg/24 h
40 wks	13-42 mg/24 h
17-Hydroxycorticosteroids	Male: 3.0-10.0 mg/24 h Female: 2.0-8.0 mg/24 h
17-Ketosteroids, total	Male: 8-20 mg/24 h Female: 6-15 mg/24 h
Osmolality	50-1400 mOsmol/kg H₂O
Oxalate	8-40 μg/mL
Potassium	Varies with diet
Proteins, total	< 150 mg/24 h
Sodium	Varies with diet
Uric acid	Varies with diet
Body Mass Index (BMI)	Adult: 19-25 kg/m²