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Protein Synthesis

What is protein synthesis?
Protein synthesis is process of producing proteins using information coded by DNA, located in the nucleus of a cell. Two processes are performed to convert the information in DNA into proteins by cells.

First, in a process called transcription, the coding region of a gene is copied to a single-stranded ribonucleic acid (RNA) version of the double-stranded DNA. This is accomplished by RNA polymerase, a large enzyme that catalyzes the linkage of nucleotides into a RNA chain using DNA as a template. The RNA is further processed into messenger RNA (mRNA) before being transported to the cytoplasm.

After processing, the mRNA is transported through nuclear pores to the cytoplasm, where translation machinery (i.e. the ribosome, eukaryotic initiation factors eIF4E and eIF4G, and poly(A)-binding protein) carry out the second process, translation, during which the ribosomes assemble amino acids in the order dictated by the mRNA sequence.

  Figure 1. The process of protein synthesis in eukaryotes.

Protein synthesis is a critical cellular process in prokaryotes and eukaryotes. This is carried out by the ribosome, an evolutionarily conserved ribonucleoprotein complex, and assisted by many other proteins and RNA molecules. Together, they synthesize all proteins needed for various biological functions. Protein synthesis can be dissected into 3 stages: initiation, elongation, and termination. Each stage has different protein and RNA molecules that play a role in efficient catalysis. The ribosome also has three main sites: the acceptor site (A site), the peptidyl-transfer site (P site) and the exit site (E site) which house tRNA, facilitating catalysis.

Initiation
Initiation begins with the 30S subunit that has initiation factor 3 (IF-3) bound to it. IF-3 binding prevents the premature binding of the 50S unit, and also plays a role in directing the mRNA strand. mRNA binds to this complex, assisted by the Shine-Dalgarno sequence. This sequence is a string of 9 nucleotide bases upstream of the start codon AUG on the mRNA. It is complementary to a sequence on the 16S rRNA of the 30S subunit and helps align the mRNA with the 30S. Next, IF-1 binds to the A site on the 30S which is the site where all charged tRNAs are first bound. IF-1 effectively blocks the premature binding of a tRNA on the A site before the ribosome has fully assembled.

IF-2 delivers the first tRNA to the P site, the site where peptidyl transfer reactions take place. In bacteria, the first tRNA is always a N-formyl modified methionine, encoded by an AUG start codon. The formyl group is removed downstream once more amino acids have been added to the nascent peptide chain. At this stage, the 30S pre-initiation complex is fully assembled which attracts the 50S subunit to spontaneously assemble with it. IF-2 is a GTP-binding protein, and hydrolysis of the GTP releases all initiation factors from the freshly assembled initiation complex. The 70S-mRNA-f-met tRNA complex is now ready for protein synthesis.

Elongation
After the 70S complex has assembled with the initiator tRNA in the P site, the ribosome starts scanning the mRNA sequence. Each codon corresponds to a particular amino acid, and this is delivered to the ribosome by elongation factor thermo unstable (EF-Tu). EF-Tu forms a complex with a charged tRNA molecule, places it onto the mRNA and then disassociates from 70S through GTP hydrolysis.

The GTP bound state of EF-Tu is essential for efficient tRNA delivery, so the cell has evolved a mechanism to recycle EF-Tu by using another protein called elongation factor thermo stable (EF-Ts). EF-Ts serves the role of a guanine nucleotide exchange factor, effectively releasing GDP from EF-Tu such that a new molecule of GTP can be bound. When EF-Tu binds another molecule of GTP, it can once again form a tRNA-EF-Tu-GTP complex and continue the tRNA delivery process. Once the A site and P site both have charged tRNAs present, a peptide bond is formed between the two amino acids through a nucleophilic attack of the A site amino acid onto the P site amino acid. At this stage, the A site contains the tRNA with the growing peptide chain and the P site has an empty tRNA.

Another GTP binding protein, elongation factor G (EF-G), catalyzes the movement of the tRNAs along the assembly line. This is called a translocation, and empties up the A site for further peptidyl transfer reactions. Once EF-G binds to the ribosome, GTP hydrolysis causes a conformational shift of the ribosome such that the tRNAs move down from the A and P site to the P and E site. The E site is the exit site and empty tRNAs difuse back into the cytosol where they are recharged by tRNA synthetases. After EF-G has carried out a translocation, the A site is ready to accept a new tRNA. Thus, the elongation cycle continues to furnish a growing nascent peptide, until a stop codon is encountered.

Termination
Once a stop codon is reached on the mRNA strand, there are no tRNA molecules that can complementary base pair with the mRNA anymore. Instead, release factors 1 and 2 (RF-1/RF-2) recognize the stop codons and bind to the 70S. This triggers the hydrolysis of the peptide chain in the P-site and releases the peptide into the cytosol for further processing and folding. RF-3, a GTP-binding protein, binds to the 70S and triggers the release of RF-1/RF-2 through GTP hydrolysis. At this stage, the 70S ribosome has the mRNA and empty tRNA bound. In this state, the 70S cannot carry out protein synthesis and must thus be recycled. This function is carried out in by ribosome recycling factor (RRF) and EF-G, which bind to the ribosome and cause its disassociation through GTP hydrolysis. Once the 30S and 50S subunits are free, IF- 3 rebinds 30S to prevent premature 70S formation and the initiation cycle can start over.

Inhibitors of protein synthesis
The sheer structural complexity of the ribosome, along with its central biological function, makes it a prime target for inhibition. Given the differences between prokaryotic 70S ribosomes and eukaryotic 80S ribosomes, organisms have evolved small molecules that can selectively target 70S ribosomes and 80S ribosomes to selectively kill off its target. These inhibitors target almost every stage of protein synthesis, with modern x-ray crystallography giving us a comprehensive understanding of their binding modes and mechanisms of action. Many 70S inhibitors serve as potent antibiotics in the clinic since they display selective toxicity towards bacterial cells. It should be noted that many inhibitors inhibit multiple steps of protein synthesis, augmenting their antimicrobial activity. Some of the key inhibitors of protein synthesis in prokaryotes are discussed below.


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