Structural Biochemistry/Nucleic Acid/Translation

In genetics, translation is the process by which mRNA is decoded and translated to produce a polypeptide sequence, otherwise known as a protein. This process is preceded by the transcription of DNA to RNA. This method of synthesizing proteins is directed by RNA and accomplished with the help of Ribosome. In translation, a cell decodes the mRNA's genetic message and assembles the brand new polypeptide chain accordingly. This genetic message is composed of a sequence of codons which comprise the mRNA strand. The translator of this message is the transfer RNA, or tRNA. The main function of tRNA is to transfer free amino acids from the cytoplasm to a ribosome, where it is attached to the growing polypeptide chain. Normally, a cell is well stocked with all 20 amino acids, either by producing them or getting them from the food we eat. The ribosome adds on the amino acids that are brought to it by tRNA molecules to the growing end of the polypeptide chain.

The Ribosome

Molecular Picture of a ribosome, Blue = Proteins, Orange = RNA, Red = Active Site

Ribosomes help coordinate the binding of tRNA anticodons with mRNA codons during translation. Ribosome can be like molecular machine that can decode RNA and use the information to build a polypeptide that contains a precise sequence of amino acid. The decoding process of the ribosome may be compared to a language translation machine that converts one language to another. So, the ribosome can be viewed as translating the language of the mRNA code into sensible protein sequences that conduct the activites of the cell. In eukaryotes, the ribosomal subunits are produced in the nucleolus of the cell. A ribosome is made up of two subunits: the large and small subunits. These subunits are made up of proteins and RNA molecules called ribosomal RNA (rRNA). The rRNA in prokaryotes and eukaryotes differ in many ways. For example, eukaryotic large subunits have a size of 60S, while prokaryotes have a size of 50S, meaning that prokaryotic subunits are smaller. Together, the large 50S and the small 30S subunit can form the 70S ribosome. The 30S subunit agrees to the selection of cognate aminoacyl tRNAs by facilitating base-pairing between mRNA codons and tRNA anticodons, while the active site or the PTC exists in the 50S subunit. The typical E. coli cell has approximately 18,000 ribosomes. The PTC catalyzes both the peptidyl transfer during protein elongation and hydrolysis of the petidyl tRNA during termination. In vitro, the 50S subunit can synthesize peptide bonds rapidly as the entire 70S ribosome, indicating that there is a catalyst. In human health, ribosome can serve as a medicinal target for drugs that work by inhibiting translation within pathogens without affecting the host organism. This is medically important because antibiotics such streptomycin and kanamycin targets the smaller subunits of bacteria while leaving the larger eukaryotic subunits unharmed.

The structure of the ribosome consists of a binding site for mRNA and three binding sites for tRNA. For tRNA, the P site (peptidyl-tRNA site) carries the growing polypeptide chain, while the A site (aminoacyl-tRNA site) holds the tRNA that carries the next amino acid that is to be added to the growing chain. The E site (exit site) is the site where discharged tRNAs leave the ribosome. The ribosome holds these two components close together and positions the new amino acid in a way that allows the addition of new amino acids to the carboxyl end of the growing polypeptide chain. As the chain gets longer, it goes through an exit tunnel in the large ribosomal subunit. Once the chain is completed, it is released to the cytosol of the cell through the exit tunnel. A particularly iconic video of Protein Translation shows this process.

Translation

In translation, there are three main steps that describe the protein decoding and synthesis process. These steps, in order, are called initiation, elongation, and termination.

Initiation

The first step of translation is called initiation. In this step, mRNA, a tRNA containing the first amino acid of the polypeptide, and two ribosomal subunits come together to start the process. The small subunit then binds to both mRNA and a specific initiator tRNA, which contains the amino acid methionine (MET). Next, the subunit scans along the mRNA strand until it reachs the start codon AUG, which indicates the start of translation process. The start codon also establishes the reading frame for the mRNA strand, with is crucial to synthesizing the protein. A shift in the reading frame results in mistranslation of the mRNA. Then, the tRNA initiator then binds to the start codon via hydrogen bonding.

The complex of consisting of mRNA, initiator tRNA, and the small ribosomal subunit attaches to the large ribosomal subunit, which completes the initiation complex. These components are brought together by the help of proteins called initiation factors which bind to the small ribosomal subunit during initiation. In addition, the cell spends GTP energy to help form the initiation complex. Once the formation of the initiation complex is complete, the initiator tRNA attaches to the P site of the ribosome, and the empty A site is ready for the next aminoacyl tRNA. The polypeptide is always synthesized in one direction, which is from the N-terminus to the C-terminus direction.

In bacteria, initiation of protein synthesis is started by binding of proteins called initiation factors (IFs) to the small 30S unit. Followed by the binding of fMet-tRNA to it. (fmet is the Met with formamyl (HCOO-) added to the N-terminus which is later removed). The three small proteins called initiation factors (IF1, IF2, and IF3) are required for the initiation of protein synthesis in bacteria. IF3 first brings mRNA and the 30S ribosome subunits together which allow the ribosome-binding site to find its complementary site on the 16S rRNA. Then, IF1 binds to and blocks the A site. The IF2 bound to GTP then escorts the initiator N-formylmethionyl-tRNA to the start codon located at the P site. IF3 is released when the initiator tRNA is in place. The 50S subunit then docks to the 30S subunit, GTP is hydrolyzed and IF1 and IF2 are released as well. This complex of Ifs-30S-fmet-tRNA now binds to the mRNA to be translated. To establish the reading frame, the mRNA in bacteria has an 8 nucleotide, purine-rich sequence called the Shine-Delgarno sequence, which is complementary to the rRNA of the small (30S) unit and helps in the initial binding of mRNA to the 30S unit complex. The Shine-Delgarno sequence is very close to the AUG codon, where the start of the translation occurs. The Shine-Delgarno sequence is present in all the bacteria.

The process of initiation of translation is complete when the large (50S) subunit binds to the Ifs-30S-fmet-tRNA complex. The binding of 50S subunit is an energy requiring process. First the energy rich GTP binds to another protein IF2 (initiation factor 2), forming the GDP-IF2 complex. The GDP-IF2 complex participates in the formation the final protein synthesizing 70S complex. In eukaryotes, additional initiation factors (IFs) are involved.

Elongation

Elongation begins with the aminoactyl tRNA that is delivered to the A site as a ternary complex with the elongation factor. The anticodon of an incoming tRNA pairs the bases with the complementary bases of the mRNA codon at the A site. The tRNA is attached to the amino acid which the mRNA codon codes for. The code which relates the codon to the amino acid is known as the Genetic Code. During this process, an elongation factor (as well as EF-Tu in bacteria) is necessary. The hydrolysis of molecules of GTP to GDP is required for codon recognition with the release of PP_i that allows for the accuracy and efficiency of the process of recognizing codons. Next, a new peptide bond is formed between the new amino acid in the A site and the carboxyl group of the growing polypeptide. This peptide formation is carried on by the help of rRNA molecule of the large subunit. Now, the peptide chain has been elongated by one amino acid. The tRNA carrying the elongated polypeptide chain is then moved to the P site while the empty tRNA in the P site is moved to the E side and exits. The process is repeated for the next incoming tRNA and amino acid: tRNA carries the next amino acid to attach to the A site, base-pairs, need energy from the hydrolysis of GTP to GDP, peptide bond formation to connect new amino acid to polypeptide chain, and appropriate movements occur, empty tRNA moves and exist to go back for another "trip", tRNA carrying elongated polypeptide chain moved to P site where it waits for another coming tRNA to take place. Therefore, one by one, an amino acid is added to the preceding amino acid.

The release factors recognize and bind to the A-site stop codon and activate the hydrolysis and release of the polypeptide from the P-site tRNA. the C terminus of the polypeptide chain attached to the P-stie tRNA that undergoes the attack at the ester carbon by a water molecule; therefore, releasing the newly synthesized polypeptide. The ribosome PTC catalyzes the aminolysis of the ester bond, where the alpha amino group of the A-site aminoacyl tRNA attacks the P-site peptidyl tRNA at the carbonyl group. this happens because the amines react faster with esters to form peptide bonds.

Note: The P site is called as such because it only binds to Peptidyl-tRNA molecule, the tRNA anchoring the growing polypeptide chain. The A Site is called as such because it only binds to incoming Aminoacyl-tRNA molecules, the tRNA that contains the free amino acid.

Termination

The elongation process stops when any stop codon, a codon signaling the end of translation, reaches the A site of the ribosome. The A site will not accept any incoming tRNA, leaving it free to bind to the release factor. The release factor will hydrolyze the bond between tRNA and polypeptide in the P site, releasing the polypeptide chain. Subsequently, the two ribosomal subunits, release factor, and mRNA come apart when their jobs are done. The polypeptide is released from the mRNA template and allowed to fold into its final 3D conformation. Also, the ribosome arrives at the end of the coding region, not the end of the RNA. So, the end of the coding region is marked by one of the three stop codons. The formation of the last peptide bond and the subsequent translocation of mRNA leads to ejection of tRNA in the E site and also brings the stop codon into the A site.

In bacteria, they have 2 class I release factors that decode the three stop codons: UAG, UAA, and UGA. The class II release factor starts the dissociation of the class I release factor from the post-termination ribosomal complex after peptidyl tRNA hydrolysis. in eukaryotes and archea, they have one class I release factor that can decode all three stop codons.

Stop Codon	Sequence
RNA (DNA)	U(T)AA
RNA (DNA)	U(T)AG
RNA (DNA)	U(T)GA

Eukaryote-specific Ribosomal Features

Microscopy studies have shown that eukaryotic 40S and 60S ribosomal subunits are largely analogous to prokaryotic 30S and 50S ribosomal subunits. Indeed, a great many structural "landmarks" are conserved, including a central protuberance, two stalks, and a sarcin-ricin loop (SRL). However, within eukaryotic and archaeal ribosomes, subunits have underwent remodeling within specific regions (the 40S subunit, for example, is divided into the "head", "beak", "platform", "body", "shoulder", "left foot", and "right foot" regions). Remodeling through protein addition has occurred primarily on solvent-exposed faces of both the 40S and 60S eukaryotic subunits. With respect to the 40S subunit, rRNA expansion segments (ESs) have networked with a variety of eukaryote-specific protein components, resulting in structural linkage atop the subunit. Similarly, the solvent-exposed face of the 60S subunit possesses two expanded regions, each containing high concentrations of ESs and eukaryote-specific protein elements.

Tertiary contact within the eukaryote-specific ribosomal proteins is essential in producing stable subunit configurations. Such proteins and their extensions are responsible for interconnection that occurs within both subunits. Within the 40S subunit, the presence of extensions rpS10, rpS12, rpS21, and rpS7 serve to link 11 proteins, creating a "daisy-chain" structure. Likewise, protein-modulated subunit connections are quite prevalent, with eukaryote-specific extensions forming networks of interaction. This produces a number of structural effects, including the presence of β sheets and α helices, which may extend tangentially across the ribosomal subunits to interact with distant regions of the structure.

Differences between prokaryotic translation and eukaryotic translation

The picture of prokaryotic translation.

The picture of eukaryotic translation.

•In general, ribosomes involved in eukaryotic translation are about 30% larger than their prokaryotic counterparts.

•Relative to prokaryotic ribosomes, eukaryotic ribosomes require a very large number of assembly, maturation, and initiation factors. They are also subjected to a great degree of regulation. While prokaryotic ribosome assembly and translational initiation are influenced of a handful of nonribosomal factors, eukaryotic ribosome development and translational initiation are modulated by approximately 200 maturation factors and a minimum of nine initiation factors, respectively.

•The P Site in prokaryotic translation is directly on AUG at the beginning, but in eukaryotic translation the ribosomal subunit scans the chain until reaches to AUG.

•Prokaryotic translation is initiated by the presence of the Shine-Dalgarno (SD) sequence, a short series of base pairs that identifies and binds to an anti-SD sequence located at the end of a 16S rRNA subunit within the ribosome. On the other hand, eukaryotic translation does not involve Shine-Dalgarno (SD) sequences, relying instead on Poly-A-Binding Protein (PABP) within a "scanning mechanism" of sorts.

•The initiating amino acid in eukaryotic translation is methionine. Conversely, the initiating amino acid in prokaryotic translation is N-Formylmethionine (fMet).

Increasing the Rate of Translation

The majority of proteins are synthesized in 20 seconds to a few minutes. This can be sped up through a number of different processes.

•A single mRNA can be bound by multiple ribosomes, with each ribosome synthesizing its own protein.

•Ribosomal subunits can be rapidly recycled by the cell. Once the ribosome has reached a stop codon down the 3' end of the mRNA strand, the subunits can reattach to the beginning to immediately begin synthesis of a new protein.

•Polysomes: polysomes are a cluster of ribosomes bound to a circular mRNA molecule. Two proteins are bound to the 5'-CAP and Poly A tail to make the mRNA circular. EIF4 binds to the 5-CAP, and Poly A Binding Protein I (PABPI) binds to the poly A tail. These two proteins associate together to make the mRNA circular, which contributes towards a more efficient translation. The ribosomes in a polysome can travel down from the 5' end to the 3' end, and only have a short distance jump to recycle back to the beginning of the 5' end of the mRNA strand and repeat translation.

Nonsense Mutations in Translation

A nonsense mutation is a point mutation (single base substitution/single nucleotide mutation) in a DNA sequence that introduces a premature stop codon in the sequence. For example, if a codon with the sequence 5' U A C 3', which codes for the amino acid tyrosine had a nonsense mutation where the C was mutated into a G, the new codon would be 5' U A G 3', which codes for a STOP codon, truncating the protein. If a nonsense mutation occurs, the cell can respond with Nonsense Mediated Decay.

Nonsense mediated decay degrades mRNA that have a nonsense mutation. The mRNA will initially undergo one round of translation, where the cell will recognize something is wrong due to the premature stop codon, and degrade the mRNA. The nonsense mutation is identified through the exon junction complexes located along the mRNA.

Exon junction complexes are simply proteins that physically associate where 2 exons come together. During translation, as the ribosome moves down the mRNA strand, exon junction complexes are "bumped" off as they come in contact with the ribosome. If translation is stopped early due to a premature stop codon, the exon junction complexes will remain on the mRNA and not be removed. This signals the cell that something is wrong, and nonsense mediated decay is allowed to degrade the mRNA.

Nonsense mediated decays are not necessarily always beneficial. It does not check to see whether or not the truncated protein is functional, but rather only looks for nonsense mutations along the strand. One such example can be seen in the disease Cystic Fibrosis.

Cystic Fibrosis occurs due to a nonsense mutation in the gene "cystic fibrosis transmembrane conductance regulator," or CFTR, which codes for a chloride channel. People without cystic fibrosis have two working copies of the CFTR gene, and only one is needed to prevent the disease. A CFTR nonsense mutation produces a truncated protein, and the cell performs nonsense mediated decay in response, degrading the CFTR mRNA. The degradation of CFTR mRNA results in very little to no CFTR channels, causing cystic fibrosis. However, scientists have discovered that the truncated protein produced due to the premature stop codon is still functional as a CFTR channel. In this case, a response designed to protect the body ultimately ends up hurting it.

References

Slonczewski, Joan L. Microbiology. "An Evolving Science." Second Edition. Klinge, Sebastian; Voigts-Hoffmann, Felix; Leibundgut, Marc; Ban, Nenad. "Atomic structures of the eukaryotic ribosome." Trends in biochemical sciences doi:10.1016/j.tibs.2012.02.007 (volume 37 issue 5 pp.189 - 198)