In this article we will discuss about the introduction and mechanisms of translation in prokaryotes.
Introduction to Translation in Prokaryotes:
The process by which proteins are produced with amino acid sequences specified by the sequence of codons in messenger RNA is called translation. Translation is the first stage of protein biosynthesis.
The main points about translation in prokaryotes are given below:
1. Site:
Translation occurs in the cytoplasm where the ribosomes are located. Ribosomes are made of a small and large subunit which surrounds the mRNA. In prokaryotic translation 70S ribosomes with 30S and 50S subunits are used. The mRNA is synthesized from DNA only. In prokaryotes, there are several initiation and termination sites.
2. Template:
In translation, messenger RNA (mRNA) is decoded to produce a specific polypeptide according to the rules specified by the genetic code. This uses an mRNA sequence as a template to guide the synthesis of a chain of amino acids that form a protein. Many types of transcribed RNA, such as transfer RNA, ribosomal RNA, and small nuclear RNA are not necessarily translated into an amino acid sequence.
3. Requirements:
The translation process requires mRNA, rRNA, ribosomes, 20 kinds of amino acids and their specific tRNAs.
4. Factors Involved:
In prokaryotes, three factors are involved in the initiation of translation [IF 1, IF 2 and IF 3], one factor in the elongation of polypeptide chain and three factors in chain termination [RF1, RF2 and RF3],
5. Enzymes Involved:
Two types of enzymes are used in translation. Aminoacyl tRNA synthetase (an enzyme) catalyzes the bonding between specific tRNAs and the amino acids. The enzyme peptidyl transferase connects A site and P site by forming a peptide bond [the nitrogen carbon bond] during elongation phase.
6. Codons Involved:
In the process of translation two types of codons, viz., start codon and stop codons are involved. The codon, AUG, initiates the process of translation and one of three stop codons i.e. UAA, UAG or UGA is used for chain termination.
7. Starting Amino Acid:
In prokaryotes, starting amino acid is N-formyl methionine. Moreover, there is overlapping of transcription and translation.
Mechanism of Translation in Prokaryotes:
Translation process consists of three major phases or stages, viz:
(1) Initiation,
(2) Elongation and
(3) Termination.
These are briefly discussed below:
1. Initiation:
This is the first phase of translation. Start or initiation codon [AUG] is responsible for initiation of translation process.
Initiation of translation in prokaryotes involves the assembly of the components of the translation system which are: the two ribosomal subunits (small and large), the mRNA to be translated, the first (formyl) aminoacyl tRNA (the tRNA charged with the first amino acid), GTP (as a source of energy), and three initiation factors (IF 1, IF 2 and IF 3) which help the assembly of the initiation complex.
The ribosome consists of three sites, the A site, the P site, and the E site. The A site is the point of entry for the aminoacyl tRNA (except for the first aminoacyl tRNA, fMet-tRNAfMet, which enters at the P site). The P site is where the peptidyl tRNA is formed in the ribosome. And the E site which is the exit site of the now uncharged tRNA after it gives its amino acid to the growing peptide chain.
Translation begins with the binding of the small ribosomal subunit to a specific sequence on the mRNA chain. Initiation of translation begins with the 50S and 30S ribosomal subunits. IF1 (initiation factor 1) blocks the A site to ensure that the IMet-tRNA can bind only to the P site and that no other aminoacyl-tRNA can bind in the A site during initiation, while IF3 blocks the E site and prevents the two subunits from associating.
IF2 is a small GTPase which binds fmet-tRNAfMet and helps its binding with the small ribosomal subunit. The 3′ end of the 16S rRNA of the small 30S ribosomal subunit recognizes the ribosomal binding site on the mRNA (Shine-Dalgarno sequence or SD), through its anti-SD sequence, 5-10 base pairs upstream of the start codon. The Shine-Dalgarno sequence is found only in prokaryotes.
This helps to correctly position the ribosome onto the mRNA so that the P site is directly on the AUG initiation codon. IF3 helps to position fMet-tRNAfmet into the P site, such that fMet-tRNAf met interacts via base pairing with the mRNA initiation codon (AUG). Initiation ends as the large ribosomal subunit joins the complex causing the dissociation of initiation factors.
The small subunit binds via complementary base pairing between one of its internal subunits and the ribosome binding site. This site a sequence of about ten nucleotides on the mRNA. It is located anywhere from 5 and 11 nucleotides from the initiating codon [AUG],
After binding of the small subunit, a special tRNA molecule, called N-formyl methionine, or fMet, recognizes and binds to the initiator codon. Then the large subunit binds resulting in the formation of the initiation complex. As soon as the initiation complex is formed, the fMet-tRNA occupies the P site of the ribosome and the A site is left empty.
This entire initiation process is facilitated by extra proteins, called initiation factors that help with the binding of ribosomal subunits and tRNA to the mRNA chain.
2. Elongation:
This is the second phase or middle phase of translation. Elongation begins after the formation of the initiation complex. Elongation of the polypeptide chain involves addition of amino acids to the carboxyl end of the growing chain. The growing protein exits the ribosome through the polypeptide exit tunnel in the large subunit.
Elongation starts when the fmet-tRNA enters the P site, causing a conformational change which opens the A site for the new aminoacyl-tRNA to bind. This binding is facilitated by elongation factor-T4 (EF-T4), a small GTPase. Now the P site contains the beginning of the peptide chain of the protein to be encoded and the A site has the next amino acid to be added to the peptide chain.
The growing polypeptide connected to the tRNA in the P site is detached from the tRNA in the P site and a peptide bond is formed between the last amino acids of the polypeptide and the amino acid still attached to the tRNA in the A site.
This process, known as peptide bond formation, is catalyzed by a ribozyme, peptidyltransferase, an activity intrinsic to the 23S ribosomal RNA in the 50S ribosomal subunit. Now, the A site has newly formed peptide, while the P site has an unloaded tRNA (tRNA with no amino acids). In the final stage of elongation, translocation, the ribosome moves 3 nucleotides towards the 3′ end of mRNA.
Since tRNAs are linked to mRNA by codon-anticodon base-pairing, tRNAs move relative to the ribosome taking the nascent polypeptide from the A site to the P site and moving the uncharged tRNA to the E exit site.
This process is catalyzed by elongation factor G (EF-G). The ribosome continues to translate the remaining codons on the mRNA as more aminoacyl-tRNA binds to the A site, until the ribosome reaches a stop codon on mRNA(UAA, UGA, or UAG).
When the A site opens again, the next appropriate aminoacyl tRNA can bind there and the same reaction takes place, yielding a three-amino acid peptide chain. This process repeats, creating a polypeptide chain in the P site of the ribosome. A single ribosome can translate 60 nucleotides per second. This speed can be vastly augmented when ribosomes unite together to form polyribosomes.
3. Termination:
This is the last phase of translation. Termination occurs when one of the three termination codons moves into the A site. These codons are not recognized by any tRNAs. Instead, they are recognized by proteins called release factors, namely RF1 (recognizing the UAA and UAG stop codons) or RF2 (recognizing the UAA and UGA stop codons).
These factors trigger the hydrolysis of the ester bond in peptidyl-tRNA and the release of the newly synthesized protein from the ribosome. A third release factor RF-3 catalyzes the release of RF-1 and RF-2 at the end of the termination process.