Translation in prokaryotes

The three basic stages of protein synthesis are initiation, elongation, termination. These process is almost similar in prokaryotes and eukaryotes.

Initiation in prokaryotes

This involves mRNA molecule, a ribosome, a specific initiator tRNA, Initiation factors(proteins) GTP and magnesium ions.

Steps involved:

  • ·       Binding of 30Ssmall ribosomal unit to the region of the mRNA with AUG initiation codon.

Actually it binds to a sequence upstream of AUG which is known as Ribosome binding site(RBS).Most of RBS are 8 to 12 nucleotides upstream from initiation codon.  This is a purine rich region almost similar to AGGAG and complementary to a pyrimidine rich region (UCCUCC) at 3’ end of 16SrRNA. RBS  was evident from work of John Shine and Lynn Dalgarno.  So this region is also known as Shine- Dalgarno sequence. Mutation occur to Shine- Dalgarno sequence or sequence complementary to it, mRNA translation will be abolished.

·         The ribosomal subunit comes to the mRNA bound to three initiation factors ( IF1, IF2, IF3)  and to a molecule of GTP and magnesium ions.

·         The next step is the binding of the initiator tRNA to the AUG start codon to which the 30S subunit is bound.

In both prokaryotes and eukaryotes, the AUG initiator codon is methionine.

·         In prokaryotes, initiator methionine is modified to form  formylmethionine(fMet) by  adding formyl group to methionine amino  group.

·         fMet attached to special initiator tRNA (tRNA. fMet) and bought to ribosome which as anticodon attached to AUG start codon.

tRNA (tRNA. fMet)is special ,because it involved specifically with the initiation process of protein synthesis.

Aminoacylation of  tRNA. fMet is as follows:

Methyionyl-tRNA  synthetase catalyzes the addition of methionine to the tRNA.

Enzyme transformylase adds the formyl group to the methionine. Thus the molecule is designated as fMet- tRNA. fMet.

·         fMet- tRNA  binds to the start codon of 30S –mRNA complex and IF3 is released and forms 30S initiation complex.

 

·         30S initiation complex consist of mRNA, 30S subunit, fMet- tRNA ,IF1 & IF2

70S ribosomes has 3 binding sites for aminoacyl tRNA:

 1. Exit(E)

2. Peptidyl (P)

3. Aminoacyl(A)

·         fMet- tRNA  is bound  to the mRNA in the P site  to form 70 S initiation complex where as E and A sites are vacant.

Elongation in prokaryotes

In prokaryotes, addition of amino acids to growing polypeptide chain one by one.This phase as 3 steps :

Aminoacyl-tRNA binds to the ribosome

The peptide bond forms

The ribosome moves along the mRNA one codon at a time(Translocation).

 Binding of Aminoacyl-tRNA

At the start of elongation, the anticodon of fMet- tRNA   is hydrogen bonded to the AUG initiation codon in P site of ribosome.

Next, the appropriate aminoacyl-tRNA binds to the codon in the A site.

Aminoacyl-tRNA  which is bound to protein elongation factor EF-Tu  and a molecule GTP is bought to ribosome.

When aminoacyl-tRNA binds to codon A,GTP hydrolysis  releases EF-Tu  -GDP. Then second elongation factor EF-Ts, binds to EF-Tu  and displaces GDP.

Next, GDP binds to EF-Tu - EF-Ts complex to produce EF-Tu –GDP complex simultaneously release of EF-Ts.

An aminoacyl-tRNA binds to the EF-Tu –GDP and that complex can bind to the A site in the ribosome.

Peptide bond formation

There are 2 steps involved in peptide formation.

·         First bond between the amino acid and the tRNA in the P site is cleaved.

·         Second, the peptide bond is formed between  by peptidyl transferase.

·         Once peptide bond has formed ,a tRNA without an attached amino acid is left in the P site.

·         The tRNA in the A site (peptidyl-tRNA )has  the first 2 amino acids of the polypeptide chain attached to it.

Peptidyl transferase activity was inhibited by antibiotics such as chloramphenicol, carbomycin.

Translocation

Ribosome  moves along the mRNA towards the 3’end.In prokaryotes ,translocation requires the activity of protein elongation factor EF-G.

An EF-G-GTP complex binds to the ribosome , GTP is hydrolyzed and translocation of the ribosome  occurs along with displacement of the uncharged tRNA away from the P site.

GTP hydrolysis changes the structure of EF-G, which facilitates the translocation event.

50Ssubunit site called E is involved in the release of the uncharged tRNA from E.coli ribosome.

The uncharged tRNA moves from the P site and then binds to the E site, effectively blocking the next aminoacyl tRNA from biinding to the A site until translocation is complete and the peptidyl-tRNA is bound correctly in the P site.

After translocation ,the EF-G is released

After translocation is complete ,A site is vacant.An amino acyl tRNA with the correct anticodon binds to the newly exposed codon  in the A site.

The whole process is repeated until translation terminate at  a stop codon.

Termination in prokaryotes

Termination of translation is signaled by any of stop codons UAG, UAA, UGA. The stop codons do not code for amino acids, so no tRNA s in the cell have anticodons

The ribosome recognizes a stop codon with the help of proteins called termination factors or release factors which  read the codons and initiate  a series of specific termination events.

 E.coli has 3 RFs (RF1,RF2,RF3) and each is a polypeptide. Factor RF1recognizes UAA and UAG.

 Factor RF2 recognizes UAA and UGA. Factor RF3 does not recognize any stop codons but stimulates termination events.

Main events are:

Release of the polypeptide from the tRNA in the P site of the ribosome  in a reaction catalyzed by peptidyl transferase

Release of tRNA from ribosome

Dissociation of the two ribosomal subunits and the RF from mRNA

The initiating amino acid  usually cleaved from the completed polypeptide.


Comments

Post a Comment

Popular posts from this blog

Biological databases

 Biological databases A database is a computerized archive used to store and organize data in such a way that information can be retrieved easily via a variety of search criteria. Databases are composed of computer hardware and software for data management. The chief objective of the development of a database is to organize data in a set of structured records to enable easy retrieval of information. Each record, also called an entry, should contain a number of fields that hold the actual data items, for example, fields for names, phone numbers, addresses, dates. To retrieve a particular record from the database, a user can specify a particular piece of information, called value , to be found in a particular field and expect the computer to retrieve the whole data record. This process is called making a query. Biological databases are  libraries of biological sciences, collected from scientific experiments, published literature, high-throughput experiment technology, and computational
Read more

Bovine Spongiform Encephalopathy (BSE)

Image
  Bovine Spongiform Encephalopathy (BSE) Spongiform encephalopathies (SEs) are degenerative disorders of the brain that occur in a number of species. They are recognized by the clinical appearance of the affected animal and the characteristic histological changes they produce in the brain.  One of the common Spongiform encephalopathy is Bovine Spongiform Encephalopathy (BSE),which mainly affect cattle. Bovine Spongiform Encephalopathy (BSE), widely referred to as “mad cow disease,” is a progressive and fatal neurologic disease of cattle. It is caused by  an unconventional transmissible agent, an abnormal prion protein . "Bovine" means that the disease affects cows, "spongiform" refers to the way the brain from a sick cow looks spongy under a microscope, and "encephalopathy" indicates that it is a disease of the brain. The infection spreads to cattle by the practice of feeding them abattoir waste and offal which may have contained scrapie infected meat.
Read more

Kirby – Bauer disc diffusion method

Aim To determine the antibiotic sensitivity of the given organism by Kirby – Bauer disc diffusion method. Principle Kirby – Bauer disc diffusion method is commonly employed for antibiotic sensitivity test. The test is based on the fact that for a given antibiotic, the size of zone of inhibition is related to MIC                  {Minimum   Inhibitory Concentration }. MIC is referred as the lowest concentration of the antibiotic that exhibits the zone of inhibition on the assay plate. Kirby – Bauer Agar disk diffusion method provides qualitative interpretive category results of susceptible, intermediate, and resistant bacterial isolate Materials required ·          Sterile forceps ·          Sterile swabs ·          Broth culture of the organism ·          Sterile Mueller-Hinton agar plates ·          Antibiotic discs   Procedure Preparation of plates Sterile Mueller-Hinton agar [pH 7.3] was prepared and poured into plates [the depth of the medium should be a
Read more