Structural Biochemistry/Proteins/Protein Translation
Translation is the process of conversion from genetic information (codons) to protein sequence(amino acids). Translation requires some components before synthesizing proteins. The components are the mRNA,tRNA,ribosomes,amino acids,and energy.
The mRNA (messenger RNA molecule) is prepared during the Transcription. dez nuts
The synthesis has three stages: Initiation, elongation, and termination.
Synthesis begins with mRNA seeking out a small ribosome. They bind in the presence of initiation factors, and the small ribosome slides along the mRNA until it reaches a start codon (AUG).The initiation aminoacyl-tRNA complex, methionine tRNA (with the anticondon 5'-CAU-3'), base pairs with the start codon. At this point, the large ribosomal subunits joins the complex, completing the ribosome. The tRNA is in the P site at this point, because it is the only part of the growing polypeptide chain.
Universally conserved factors
Bacterial IF1 functions mainly to prevent tRNA binding to the A site, stabilize the mRNA binding by the small subunit, and stabilize the binding of fMet-tRNA/IF2/IF3 to the small subunit of the ribosome. IF1 affects IF3 by increasing IF3’s antiassociation activity and coordinates with IF2 on the ribosome for their simultaneous release after initiation procedure. eIF1A is the archeal and eukaryotic equivalent of IF1 and is identical in structure with the exception of an extra tail at the N-terminal and helix at the C-terminal, the function of which is not yet fully understood.
Universally conserved IF2 in bacteria and eIF5B in archaea are primarily involved in joining of the small and large subunits of their respective ribosomes. After subunit joining, the GTPase associated center of the large subunit causes the IF2 or eIF5B hydrolysis reaction that releases IF2/GDP or eIF5B/GDP along with IF2 or eIF1A. IF2 has been proven to help locate the fMet-tRNA in bacteria while no such process has been proven of the eIF1A in archaea or eukaryotes.
The main role of IF3 is bacterial ribosomal association and dissociation as it works along with IF1 and IF2. IF3 is also involved in locating the start codon and helping to select fMet-tRNA. The carboxyl terminal section of IF3 at sufficient concentration can perform the function of the whole IF3 shown by cleaving the protein below the carboxyl domain. It acts as a competitor to subunit association by binding to the active site of the small subunit and inducing conformational changes.
Although eIF3 is smaller and dissimilar in structure and sequence to IF3, it performs some similar functions. eIF3 is able to recognize AUG start codons and discriminate against non-AUG codons at its carboxyl end. It functions similarly to IF3 by binding to almost the site as the carboxyl domain of IF3 inducing dissimilar conformational changes. eIF3 differs from IF3 in that it does not help select Met-tRNA.
eIF2 is a heterotrimer that is involved in the selection of initiator tRNA and binding of the Met-tRNA to the small subunit. An eIF2/GTP complex forms a ternary complex with Met-tRNA by direct binding whereas eIF2 with GDP or bound to a tRNA without the methionine residue is unlikely to form a strong ternary complex and dissociation is likely. The eIF2/GTP/Met-tRNA ternary compound binds to the 40S subunit of eukaryotic ribosomes and following recognition the eIF2 is detached and recycled by hydrolysis of GTP. This GTP hydrolysis in eukaryotes requires an additional eIF5.
eIF4G is a large protein that forms the scaffold for the construction of the cap binding complex. It is also responsible for the recruitment of the 43S IC in forming the 5-prime cap that is required for efficient translation. It has also been shown the eIF4G binds to eIF3.
Once the complex has been formed, the ribosome can slide along the mRNA, adding new amino acids as it goes. Hydrogen bonds form between the mRNA codon in the A site and the complementary tRNA anticodon. This fills the A site. We now have a charged aminoacyl-tRNA n both the A site and the P site. The enzyme, peptidyl transferase, uses the energy that was stored in the aminoacyl-tRNA complex when the amino acid was loaded (from GTP) to catalyze the formation of a peptide bond. The aminoacyl-tRNA used for this is the one in the P site. Now the tRNA in the P site is free, and there is still an aminoacyl-tRNA in the A site. This aminoacyl-tRNA has its own amino acid, which is now bound to a methionine. Translocation is necessary to add the next amino acid residue. The ribosomal assembly slides in a 5' to 3' direction along the mRNA. This moves the next codon into place in the A site. At the same time, the deacylated tRNA from the P site is moved to the E site displacing the previously deacylated tRNA and the aminoacyl-tRNA that is carrying out nascent chain moved from the A site to P site. The process is ready to begin again with an empty A site.
In bacteria, the aa-tRNA is bound to GTP and elongation factor EF1A as a ternary compound before reaching the ribosome. EF1A is nonspecific and will bind to most aa-tRNAs based on varying levels of affinity for the tRNA or amino acid. Notable exceptions that have weak binding to EF1A include the intiator tRNA fMet-tRNA and Asp-tRNA. The homolog elongation factor eEF1A in eukaryotes has similar characteristics to EF1A.
Non-complementary or non-cognate aa-tRNA/GTP/EF1A complexes have an equal chance of binding to the ribosome as the correct complementary aa-tRNA ternary complex. There are two exclusion methods to ensure the correct matching for aa-tRNA complexes with the mRNA. First, conformational changes in the aa-tRNA complex and the ribosome allow for the codon and anticodon to make initial contact. Non-cognate ternary aa-tRNA complexes will dissociate quickly and GTP hydrolysis by EF1A is unlikely to occur. Base pairing is obeyed up until the third base pair and thus nearly cognate aa-tRNA complexes are excluded by the universally conserved nucleotides 530, 1492 and 1493.
After correct complementary matching of the aa-tRNA ternary complex and ribosome, the small subunit of the ribosome assumes a closed conformation that promotes GTP hydrolysis by EF1A. The second process of elimination of near cognate aa-tRNA occurs in the PTC (peptidyl transferase center). Near cognate aa-tRNA have a much lower rate of accommodation compared to rate of dissociation while cognate aa-tRNA have a very low dissociation rate compared to their association rate. These two methods of exclusion for near-cognate aa-tRNA combine to give very low percentages of mutation during elongation.
A second elongation factor, EF2/GTP attaches to the ribosome at the same site as the aa-tRNA/GTP/EF1A ternary complex and induces translocation of tRNA and mRNA one codon down. The acceptor site of the tRNA is thought to move first from the A to P site followed by the movement of the tRNA anticodon and mRNA codon with the small subunit of the ribosome rotating against the large subunit. EF1A/GDP is recycled by a guanine nucleotide exchange factor to reform EF1A/GTP while the dissociation rate of EF2/GDP is fast enough to allow EF2/GTP and EF2/GDP to exist in near equilibrium.
Translation has its own set of stop signs. If the codon in the A site is UGA, UAA, or UAG, it is known as a termination codon. Instead of a new aminoacyl-tRNA binding to the A site, a protein called released factor binds to the termination codon, causing a water molecule to be added to the polypeptide chain. The chain will then be released from the tRNA in the P site, and the two ribosomal subunits will dissociate and as well as increase the amount of protein that may be made from a single transcript, several ribosomes may translate a message at the same time. This is known as a polyribosome.
Differences Between Prokaryotes and Eukaryotes
Due to prokaryotes' significantly smaller amount of DNA, translation happens only one protein at a time. However, because prokaryotes do not have a nucleus, translation occurs at the same time as transcription. In eukaryotes, one complete strand of mRNA can be translated by many ribosomes at once, thus drastically reducing the amount of time required to produce a feasible amount of proteins, but transcription and translation are separate events. Transcription occurs in the nucleus and the mRNA is exported to the cytoplasm before translation can occur.
Also, prokaryotic ribosomes are similar in structure to eukaryotic ribosomes, but not identical. Prokaryotic ribosomes are smaller (30S for the small subunit, 30S for the large, whereas for eukaryotes, it's 40S and 60S, respectively). Thus drugs that prevent bacterial infection by stopping translation can specifically target the bacteria and leave the host cells to function normally.
Evolution of Translation in Eukaryotes
Translation in Eukaryotes is highly regulated and are regulated by universal and lineaged mechanisms. Recent discovery has yielded information that suggests that the mechanisms that regulate translation emerged at different times based on evolutionary need. The evolution of eukaryotes thus paralleled the evolution of translation. Some mechanisms evolved independently of translation but were later incorporated into it. The thinking now by scientists suggests that the mechanisms that regulate translation may have been involved in other cellular processes and were later incorporated into translation. This overall view has been dubbed by scientists as 'tinker' which involves co-opting and assembling molecules and regulatory mechanisms from other cellular processes.
Walsh, Christopher. "Posttranslational Modification of Proteins: Expanding Nature's Inventory." Roberts and Co. (2006): 2-6.
Campbell and Reece. "Biology, 8th Edition"