PROTEIN SYNTHESIS INHIBITORS

OVERVIEW

RIBOSOMAL STRUCTURE

BACTERIA -- 30S + 50S --> 70S

30S subunit

• binds mRNA in initiation
• holds growing peptide chain

50S subunit

• accepts / translocates charged tRNAs
• "A" site --> AMINOACYL-tRNA (ACCEPTOR) SITE
• "P" site --> PEPTIDYL-tRNA (DONOR) SITE

MAMMALS -- 40S + 60S --> 80S

THREE STAGES OF PEPTIDE SYNTHESIS

• INITIATION
• ELONGATION
• TERMINATION

INITIATION OF PEPTIDE SYNTHESIS

MECHANISM

IF-3 + 30S + mRNA --> mRNA-30S COMPLEX

AA-tRNA + GTP + IF-2 --> tRNA COMPLEX

• (Prokaryotes --> AA nearly always N-FORMYLMETHIONINE
• (Eukaryotes --> AA usually METHIONINE

tRNA COMPLEX + IF-1 + mRNA-30S COMPLEX -->

CODON + ANTICODON INITIATION COMPLEX

IF-1, IF-2, IF-3 REMOVED

30S INITIATION COMPLEX + 50S SUBUNIT + ENERGY -->70S RIBOSOME + GDP + Pi

DRUGS

AMINOGLYCOSIDES --

Action partially due to FREEZING the complex

[according to West & Neu, Pradhan et al 87, p759]

ELONGATION OF PEPTIDE CHAINS

MECHANISM

2nd AA-tRNA + GTP + EF-1 --> COMPLEX

COMPLEX INSERTED INTO "A" (AMINOACYL-tRNA) SITE (70S) - -> releasing GDP-EF-1 + Pi

PEPTIDYL TRANSFERASE CATALYZED alpha-AMINO NUCLEOPHILIC ATTACK ON ESTERIFIED CARBOXYL OF PEPTIDYL-tRNA (growing chain)

RESULT:

• GROWING CHAIN NOW ON "A" SITE
• "P" SITE: UNCHARGED tRNA VACATES SITE

EF-2 + GTP TRANSLOCATE GROWING CHAIN TO "P" SITE

CYCLE REPEATS

DRUG ACTIONS

PUROMYCIN -- CAUSES TERMINATION OF CHAIN

Resembles tyrosinyl-tRNA WHICH WILL TERMINATE

TETRACYCLINES --

REVERSIBLY INHIBIT AA-tRNA BINDING TO 30S SUBUNIT

WEST & NEU, PRADHAN 87, P760.

MACROLIDES

• 50S - near "P" --> CONFORMATIONAL CHANGE
• no peptide bonds formed, but ribosomal cycle continues
• Inhibit TRANSLOCATION (A --> P)
• Some mutants do NOT bind --> resistant
• G+ accum 100x G- orgs
• Macrolides-CAP-Lincomycin COMPETE for sites
  • Binds reversibly to 50S ribosome near the "P" (donor site) block binding of tRNA
  • May compete with CAP and lincomycin for site.
• Orgs accum 100x more erythromycin than do g- orgs.
• Effective only against actively dividing organisms.

LINCOSAMIDES

• 50S - near "P" SITE & INTERFERE WITH BINDING OF AMINOACYL END OF THE AA-tRNA
• OCCUPY SITE or CHANGE RIBOSOMAL CONFORMATION
• DESTABILIZES RIBOSOMES SO GROWING CHAINS "FALL OFF" mRNA
• WEST & NEU 87, PRADHAN, P 760.

CHLORAMPHENICOL

• REVERSIBLE BINDING TO 50S -- NEAR "P" SITE & INTERFERE WITH BINDING OF AMINOACYL END OF AA-tRNA
• INHIBITS PEPTIDE BOND FORMATION BETWEEN AA-tRNA & GROWING CHAIN ON "P" SITE

AMINOGLYCOSIDES -- CIDAL

• IRREVERSIBLE BINDING TO 30S SUBUNIT --> BIND TO PROTEIN P10 -->
  • DEPLETION OF RIBOSOMAL POOL
  • "STREPTOMYCIN MONOSOMES"
• (Actually an effect on initiation)
• MISREADING OF mRNA --> FAULTY PERIPLASMIC PROTEINS ACCUMULATE MEMBRANE --> MEMBRANE DAMAGE/LEAK
• INHIBITION -- RESISTANCE -- DEPENDENCE
  • RIBOSOMAL PROTEIN S12 (? P10 ?)
  • LYS -- NORMAL IN POSITION 42 (SENSITIVE)
  • ASPARAGINE -- NO STREPT. BINDING (RESIST)
  • GLUTAMINE -- NEEDS STREPT. BINDING (DEPENDENT)

Notes:

• strept = streptomycin
• WEST & NEU 87 PRADHAN, P 803 SAY STREPTOMYCIN BINDS TO PROTEIN P10. GLC OLD NOTES SAID S12.

TERMINATION OF POLYPEPTIDE CHAIN

MECHANISM

mRNA NONSENSE (TERMINATING) CODON IN "A" SITE

NO CORRESPONDING tRNA

RELEASING FACTORS RECOGNIZE TERMINATING CODON

RELEASING FACTOR + GTP + PEPTIDYL TRANSFERASE

HYDROLYZE POLYPEPTIDYL-tRNA BOND IN "P" SITE --> RELEASE POLYPEPTIDE

70S RIBOSOME --> 30S + 50S SUBUNITS

INTERESTING TIDBITS

• 1 MAMMALIAN RIBOSOME TRANSLATES 200 CODONS IN 2 MINS --> 20,000 MW PROTEIN
• "POLYSOMES" -- MULTIPLE RIBOSOMES ON SAME mRNA

DRUGS

BECAUSE OF THEIR EFFECTS, THE FOLLOWING TERMINATE PROTEIN SYNTHESIS BUT DO NOT ACT SPECIFICALLY ON THE "TERMINATION SEQUENCE" JUST OUTLINED --

• CHLORAMPHENICOL
• ERYTHROMYCIN
• LINCOMYCIN

COMPARISON OF DRUGS ON PROKARYOTIC vs EUKARYOTIC

      Drugs on Prokaryotic vs Eukaryotic Protein Synthesis        

                   ------------EUKARYOTES------ PROKARYOTES         
                             --------                               

                CYTOPLASMIC     MITOCHONDRIAL                       

INITIATION                                                          

ELONGATION                                                          

Chloramphenicol       --                +                 +         

Cycloheximide          +               --                --         

Lincomycin            --                ?                 +         

Puromycin              +                +                 +         

Tetracycline          --                +                 +         

TERMINATION                                                         

Chloramphenicol       --                +                 +         

Erythromycin          --                +                 +         

Lincomycin             ?                ?                 +         

Streptomycin           +              +/--                +         

+ = Inhibition;  - = No Inhibition;  ? = Unknown                  
[Harper's Biochemistry has a series of nice illustrations to      
facilitate study of this material. glc]                           

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