Protein Synthesis Steps In Brief
The process of protein synthesis translates the codons (nucleotide triplets) of the messenger RNA (mRNA) into the 20-symbol code of amino acids that build the polypeptide chain of the proteins. The process of mRNA translation begins from its 5′-end towards its 3′-end as the polypeptide chain is synthesized from its amino-terminal (N-end) to its carboxyl-terminal (C-end). There are almost no significant differences in the protein synthesis steps in prokaryotes and eukaryotes, however there is one major distinction between the structure of the mRNAs – prokaryotes often have several coding regions (polycistronic mRNA), while the eukaryotic mRNA has only one coding region (monocistronic mRNA).
The main protein synthesis steps are:
In most of the aspects, the process in eukaryotes follow the same simple protein synthesis steps as in prokaryotes. However there are specific differences that could be outlined. For example, one important difference is that in prokaryotic cells the process of translation starts before transcription is completed. This coupling is defined because prokaryotes have no nuclear membrane and thus there is no physical separation of the two processes.
Protein Synthesis InitiationThe first of protein synthesis steps is initiation that cover the assembly of the translation system components and precedes the formation of peptide bonds. The components involved in the first step of protein synthesis are:
- the mRNA to be translated
- the two ribosomal subunits (small and large subunits)
- the aminoacyl-tRNA which is specified by the first codon in the mRNA
- guanosine triphosphate (GTP), which provides energy for the process – eukaryotes require also adenosine triphosphate!
- initiation factors which enables the assembly of this initiation complex - prokaryotes have 3 initiation factors are known (IF-1, IF-2, and IF-3), while eukaryotes, there have over ten factors designated with eIF prefix.
- Shine-Dalgarno (SD) sequence - In Escherichia coli is observed sequence with high percentage of purine nucleotide bases, known as the Shine-Dalgarno sequence. This region is located close to 5’ end of the mRNA molecule, 6-10 bases upstream of the initiating codon. The 16S rRNA component of the small ribosomal subunit possess a complementary to the SD sequence near its 3'-end. Thus the two complementary sequences can couple, which facilitates the positioning of the 30S ribosomal subunit on the mRNA in proximity to the initiation codon. The mechanism is slightly different in eukaryotes because they do not have SD sequences. In eukaryotes, with the assistance of the eIF-4 initiation factors, the 40S ribosomal subunit binds close to a structure called “cap structure” at the 5-end of the mRNA and then moves down the messenger RNA sequence till it finds the initiating codon. However this process requires energy from ATP.
- Initiating codon (AUG) - The initiating AUG triplet is recognized by a special initiator tRNA. In prokaryotes this event is facilitated by IF-2-GTP, while in eukaryotes by eIF-2-GTP and additional eIFs. The charged initiator transport RNA aproaches the P site on the small ribosomal subunit. In bacteria (and in mitochondria), a methionine is attached to the initiator tRNA an subsequently a formyl group is added by the enzyme transformylase, which uses N10-formyl tetrahydrofolate as the carbon donor – finally a N-formylated methionine is attached to the initiator tRNA. For comparison, in eukaryotes, the initiator transport RNA attaches a non formylated methionine. In both types of cells, this N-terminal methionine attached to the 5’-end is removed before the end of the translation. In the last step of the initiation, the large ribosomal subunit joins the complex formed by now, and thus a fully functional ribosome is formed. This complex has a charged initiating tRNA in the P site, and the A site empty. During this protein synthesis step is used the energy within the GTP on (e)IF-2, which gets hydrolyzed to GDP. The reactivation of (e)IF-2-GDP is facilitated by A guanine nucleotide exchange factor.
Translation elongation is second in protein synthesis steps. During the elongation step the polypeptide chain adds amino acids to the carboxyl end the chain protein grows as the ribosome moves from the 5' -end to the 3'-end of the mRNA. In prokaryotes, the delivery of the aminoacyl-tRNA to ribosomal A site is facilitated by elongation factors EF-Tu-GTP and EF-Ts, and requires GTP hydrolysis. In eukaryotes, the analogous elongation factors are EF-1α−GTP and EF-1βγ. Both EF-Ts (in prokatyotes) and EF-1βγ (in eukaryotes) function as nucleotide exchange factors.
The peptidyl-transferase is an important enzyme which catalyzes the formation of the peptide bonds. The enzymatic activity is found to be intrinsic to the 23S rRNA found in the large ribosomal subunit. Because this rRNA catalyzes the polypeptide bound formation reaction, it is named as a ribozyme.
The transport RNA at the P site carries the polypeptide synthesized by now, while on the A site is located a tRNA, which is bound to a single amino acid. After the peptide bond has been formed between the polypeptide and the amino acid, the newly formed polypeptide is linked to the tRNA at the A site. Once this step is completed, the ribosome moves 3 nucleotides toward the 3'-end of the mRNA. This process is known as translocation - in prokaryotes, it requires the participation of EF-G-GTP and GTP hydrolysis, while the eukaryotic cells use EF-2-GTP and GTP hydrolysis again. During the translocation, the uncharged tRNA moves from the P to the E site and peptidyl-tRNA leaves the A site and go to the P site. This is an iterative process that is repeated until the ribosome reaches the termination codon.
Termination of Translation
Termination happens when the A site of the ribosome reaches one of the three termination codons (UAA, UAG or UGA).In prokaryotes, these codons are recognized by different release factors (abbreviated with RF). RF-1 is responsible for the recognition of termination codons UAA and UAG, while RF-2 - UGA and UAA. When these release factors bind the complex, this cause in hydrolysis of the bond linking the peptide to the tRNA at the P site and releases the nascent protein from the ribosome. Then a third release factor (RF-3-GTP) causes the release of RF-1 or RF-2 as GTP is hydrolyzed to GDP and single phosphate reqidue. In contrast, the eukaryote cells have just one release factor, eRF, which can recognize all three termination codons. A second factor is involved - eRF-3, with a similar function to the RF-3 in prokaryote cells. The protein synthesis steps in prokaryotes are summarized in figure below. Some antibiotic inhibitors that could be involved at different protein synthesis steps are:
- diphtheria toxin, which inactivates EF-2 and thus prevents the translocation
- clindamycin and erythromycin, which blocks (due to irreversible binding) to a site within the 50S sub-unit of the ribosome and in this way inhibit the translocation
- ricin (from castor beans) is a very potent toxin that exerts its effects by removing an adenine from 28S rRNA, thus inhibiting the function of eukaryotic ribosomes.