The proteins which are to be degraded are tagged with ubiquitin (76AA) at the lysine residue of misfolded protein with the Isopeptide bond.
6.8.6. Proteolytic Cleavage:
Generally some sequences at the N terminus function as signal peptides and undergo cleavage during post translational modification. Examples: a) post translational processing of Pre-POMC(Pro ophio melanocrtin) in the pituitary. b) A signal peptide for approx 24 aminoacids at the N terminus of Pro-Insulin. Active insulin is produced through cleavage of two peptide chains linked together through disulphide bonds. c) Zymogens are also activated by the proteolytic cleavage.
6.8.7. Intein splicing:
The non functional sequences or portions in a protein are called as Inteins and they are removed during PTM. They are protein introns. Cysteine, threonine or serine are present at the splicing junction. This is conserved throughout the evolution. They have OH and SH group respectively which act as a Nucleophile and bring about the transesterification reaction which joins the two exteins. This is a self catalysed reaction. The spliced out intein acts as a potential double standed DNA cutter and contain LAGLIDADG motif. So if this process occurs in the prokaryotic system, this intein will cut the dsDNA and will produce double stranded break which cannot be repaired and eventually lead to the death of the cell. Thats why the Inteins are considered to be selfish. But if this is occurring in the eukaryote, no harm will be there as they have the double copies of the same gene and thus the double stranded produced by the intein can be overcome easily with the double standed break repair.
DnaE is the catalytic subunit of DNA polymerase III present in cynobacterium Synechocystis sp. PCC6803, is a split gene product containing Inteins which undergo Trans-splicing to produce intact DnaE protein.
6.9. Antibiotics effect translation
Rifampicin inhibits DNA dependent RNA polymerase by binding its beta-subunit in turn inhibit prokaryotic DNA transcription. It has lipophilic nature makes it a good candidate in the treatment meningitis form of tuberculosis, which requires distribution to the central nervous system and penetration through the blood-brain barrier.
- Linezolid inhibit the formation of the initiation complex, though the mechanism is not fully understand.
- Aminoacyl tRNA entry
- Tetracyclines and Tigecycline (a glycylcycline related to tetracyclines) inhibting the binding of aminoacyl tRNAs by block the A site on the ribosome.
- Aminoglycosides, causing amplified rate of error in production of polypeptide chain by premature termination because causing hamper within the proofreading process also inhibit translocation, disrupt the integrity of bacterial cell membrane and binds with 30S ribosomal subunit.
- Kirromycin: Function of EF-Tu inhibited by an antibiotic name kirromycin. When EF-Tu is bound by kirromycin, then EF-Tu-GDP complex cannot be released from the ribosome and prevent formation of the peptide bond between the peptidyl-tRNA and the aminoacyl-tRNA. Thus arrest of translation occurs.
- Peptidyl transfer
- Chloramphenicol in both bacteria and mitochondri blocks the peptidyl transfer reaction in 50 S subunit of ribosome.
- Macrolides inhibiting peptidyl transfer as well as ribosomal translocation in 50 S subunit.
- Syneragistic act reported in case of Quinupristin/dalfopristin, with dalfopristin enhancing the binding of quinupristin to 50 S subunit and inhibiting peptidyl transfer, Quinupristin binds to a close site on the 50S ribosomal subunit and inhibiting elongation of the polypeptide simintaniouly causing incomplete chains to be released.
- Cycloheximide: It binds to 80 S ribosome and inhibit the peptidyl transferase reaction.
- Ribosomal translocation
- Clindamycin inhibition of ribosomal translocation.
- Aminoglycosides and macrolides have evidence of inhibition of ribosomal translocation.
- Fusidic acid block elongation by preventing the turnover of elongation factor G (EF-G) from the ribosome.
- Ricin: Ricin is a toxic protein extrac from the castor bean (Ricinus communis) cause depurination of a specific adenosine in 28 S RNA of 60 S subunit. Ricin is classified as a type 2 ribosome inactivating protein (RIP) which is also known as holotoxins, are heterodimeric glycoproteins Neomycin. Whereas single enzymatic protein chain present in Type 1 RIPs.
- Neomycin: It is an aminoglycoside antibiotic, which is present in various topical medications such as creams, ointments, and eyedrops and other medicinally important drugs.
- Puromycin: It has a structure similar to the tyrosinyl aminoacyl- tRNA or we can say it has structure analogue 3’ end of amino acytRNA. It binds to the ribosomal A site and participates in peptide bond formation, producing peptidyl-puromycin and its amide bond not cleave by peptidal transferase thus not engage in translocation and quickly dissociates from the ribosome causing a premature termination of polypeptide synthesis.
- Macrolides and clindamycin (both also having other potential mechanisms) responsible for premature dissociation of the peptidyl-tRNA from the ribosome.
- Streptogramins: It also responsible for premature release of the peptide chain.
The following antibiotics bind to the 50S ribosomal subunit:
Chloramphenicol: It bacteriostatic (that is, it stops bacterial growth) inhibit peptide bond formation because bind to A2451 and A2452 residues in the 23S rRNA of 50 S subunit and inhibit peptidyl transferase reaction. It directly interferes with substrate binding.
Erythromycin: It inhibits peptide chain formation in elongation by bind to 50 S subunit.
Linezolid: Linezolid binds to the 23S rRNA present in 50S subunit (peptidyl transferase activity) and inhibit protein synthesis. It site close to the binding sites of chloramphenicol, lincomycin, and other antibiotics. Due to this unique mechanism of action, cross-resistance between linezolid and other protein synthesis inhibitors is highly uncommon or absent.
Tetracycline: It binds to the 16S rRNA present in 30S ribosomal subunit and prevents the amino-acyl tRNA from binding to the A site of the ribosome and thus inhibiting cell growth. It has reversible nature of binding.
Streptomycin: It is a aminoglycosides and trisaccharide, responsible for misreading of mRNA in relatively low concentration by binding through 30 S subunit. paromomycin, kanamycin and streptomycin are aminoglycosides are known for their ability to bind to duplex RNA with high affinity.
Diptheria toxin: It is an exotoxin of Cornephage β (lysogenic phage) infected cornybecterium diptheriae, which inactivate eEF2 (E.K.) by ADP-ribosylation, A fragment of toxin responsible for ribosylation.