Introduction and Goals
Translation, the mechanism of converting mRNA sequences into protein sequences, will be described in this tutorial. The genetic code is "cracked" during the process of translation; that is, triplets of nucleotides are read and used to specify which amino acids should be added to the growing peptide. Translation occurs on the RNA-protein complex termed a ribosome. This process requires the activity of additional RNAs (the transfer RNAs that deliver the amino acids and the ribosomal RNAs that facilitate mRNA-binding and translation on the ribosome).
By the end of this tutorial you should know:
- How the nucleotide sequence in the mRNA encodes for the amino acid sequence in the protein
- The role of codons and reading frames
- The structure and function of tRNA, including the anticodon and amino acid attachment
- The structure of a ribosome
- The three phases of translation: initiation, elongation and termination
- The mechanisms of recruiting ribosomes to the ER and the insertion of a newly synthesized polypeptide across the ER membrane
The Genetic Code
Translation is the process of using the genetic code of the mRNA to determine the amino acid sequence of the protein. This is literally a translation from one language into another, from the four-letter nucleotide code to the twenty amino acids code. There is not a one-to-one correspondence between nucleotides and amino acids; rather, 3 consecutive nucleotides in the mRNA (termed codons) are translated into a single amino acid. There are 64 possible combinations of triplet nucleotides, and therefore 64 possible codons. Figure 1 is a codon table illustrating the genetic code, the codons, and the amino acids they specify. This genetic code is universal, with a few exceptions in mitochondria (which have their own genomes). Three things are worth noting in Figure 1. First, the three codons highlighted in red (UAA, UAG and UGA) do not code for amino acids; they are the stop codons that signal the termination of translation. Second, the codon AUS (highlighted in green) is referred to as the start codonbecause it is always the first codon in a protein coding sequence. Third, the genetic code is redundant, meaning some amino acids are specified by more than one codon. In fact, there are 61 codons and only 20 amino acids. Each codon only specifies a single amino acid, but a single amino acid may be specified by multiple codons. For example, the codon GCA always translates to the amino acid alanine, however, alanine can also be specified by the codons GCC, GCG and GCU.
Messenger RNA (mRNA) has Three Reading Frames
A single strand of mRNA is translated from the 5' to 3' end. It can be translated in three possible reading frames, depending on where the first triplet is positioned (illustrated in Figure 2); however, only one reading frame will encode the correct protein sequence. Using the genetic code, one can examine a sequence of mRNA (or its corresponding DNA sequence) and predict the protein sequence by "conceptually" translating in all three frames. Often if the mRNA sequence is translated in the wrong frame, there are multiple premature stop codons encountered. The reading frame that encodes the correct protein is referred to as the open reading frame. A single nucleotide insertion or deletion mutation can be devastating if it occurs in the DNA sequence encoding the open reading frame of a gene. Not only will the codon that is altered be affected, but the entire reading frame will be shifted.
Transfer RNAs (tRNAs) are the Key to the Genetic Code
The codons of mRNA do not directly recruit the correct amino acids during translation. Rather, the transfer RNAs (tRNAs) act as adaptor molecules, connecting the codon to the correct amino acid. The tRNAs are small RNA molecules (70 - 90 nucleotides) with a distinct secondary structure. This structure arises from intramolecular base pairing, resulting in three loops reminiscent of a three-leaf clover (see Figure 3). One of these loops contains the anticodon, a triplet of nucleotides that are complementary to a codon. The 3' end of the tRNA (the stalk part of the three-leaf clover) is the site of amino acid attachment. Once an amino acid is added to a tRNA, it is referred to as a charged tRNA. Adding the correct amino acid to the tRNA is critical to interpreting the genetic code. Enzymes termed aminoacyl-tRNA synthetasescovalently link the amino acid to the tRNA in an ATP-dependent reaction. The amino acid is linked to the tRNA in a high-energy bond through its carboxyl group, leaving the amino group free. There are twenty different aminoacyl-tRNA synthetases, one for each amino acid, that are capable of charging the appropriate tRNA. The charged tRNA recognizes the codon in the mRNA via its complementary anticodon and delivers the correct amino acid. Thus, the charged tRNA is the molecule that literally translates the nucleotide sequence of the mRNA into the amino acid sequence of the protein. Some tRNAs can anneal stably with a codon that is not a perfect match to its anticodon, as long as the first two of the three nucleotides in the codon are complementary. This is referred to as the third-position wobble and allows the cell to use fewer tRNA molecules to encode amino acids for all the codons. The tRNAs deliver the appropriate amino acids and they are joined together by a peptide bond.
Translation is Carried Out by the Ribosome
The recruitment of the appropriate tRNA to the codon of the mRNA requires the assembly of a large RNA-protein complex referred to as theribosome. A ribosome is composed of 50-80 different proteins and 3-4 ribosomal RNAs (rRNAs), depending on whether it is prokaryotic or eukaryotic. These proteins and rRNAs are organized into a small and a large subunit (illustrated in Figure 4). Eukaryotic ribosomes are found free in the cytoplasm or associated with the endoplasmic reticulum (ER). Despite their differences in size and composition, the ribosomes of prokaryotic and eukaryotic cells are similar in structure and function. A ribosome functions as a molecular machine that assembles the mRNA and appropriate tRNAs to synthesize the protein. A ribosome is required for peptide bond formation between the amino acids delivered by the tRNAs. The small subunit contains the mRNA binding site, and the process of translating the mRNA into amino acid sequence takes place at the interface between the small and large subunits. There are three distinct sites in a ribosome: the A site (aminoacyl-tRNA), where the incoming charged tRNA enters; the P site (peptidyl-tRNA), where the tRNA with the growing peptide chain resides; and the E site, where the tRNA (devoid of its amino acid) exits. These binding sites in a ribosome are largely formed by the rRNAs, which make up the bulk of the ribosome.
Translation can be divided into three distinct phases: initiation, elongation and termination. During initiation, the mRNA is bound by the small ribosomal subunit and the initiator tRNA that carries the amino acid methionine (complementary to the codon AUG). The initiator tRNA is unique in that it first occupies the P site, whereas all other incoming tRNAs first occupy the A site (illustrated in Figure 5). In prokaryotes, the initiator codon is coupled to a modified methionine (formylmethionine). The small subunit of the ribosome (loaded with the initiator tRNA) recognizes and binds to the 5' end of the mRNA and moves along the mRNA until the first AUG codon (start codon) is encountered, at which point the large ribosomal subunit is recruited. Translation elongation begins by the recruitment of another charged tRNA to the empty A site of the complete ribosome (small subunit + large subunit).
In prokaryotes, the small ribosomal subunit recognizes the 5' end of the mRNA through a short sequence upstream of the start codon. The ribosome-binding sequence(also called the Shine-Delgarno sequence) is complementary to one of the rRNAs within the small subunit of the ribosome. In eukaryotes, the small subunit of the ribosome recognizes and binds to the 5' cap of the mRNA. Translation initiation in eukaryotes also requires additional proteins, termed translation initiation factors, which associate with the ribosome and bind to the 5' cap, as well as the 3' poly-A tail, to ensure that the mRNA is intact. Once the small subunit of the ribosome has found the start codon, the initiation factors dissociate from the small subunit and the large subunit of the ribosome is recruited.
The process of translation elongation begins after the complete ribosome has recognized the start codon; at this point, the synthesis of a protein can begin. The ribosome moves along the mRNA in the 5' to 3' direction, starting from the AUG and translating every codon into an amino acid. Remember that when translation is initiated, the P site is occupied by the initiator tRNA and the A site is empty and ready to receive another charged tRNA. Elongation occurs in four steps (illustrated in Figure 6). First, a charged tRNA is delivered to the A site with the aid of translation elongation factorsthat bind GTP. Once the charged tRNA is delivered to the A site, GTP is hydrolyzed and the elongation factors dissociate from the ribosome. Second, the ribosome checks that the codon and anticodon are complementary; if the tRNA is incorrect, the base pairing between the anticodon of the tRNA and the codon of the mRNA will not be very stable and the tRNA will be released before a peptide bond can be formed. Third, if the correct charged tRNA is delivered, then the base paring between the anticodon of the tRNA and the codon of the mRNA will be stable and the tRNA will remain bound to the A site long enough for a peptide bond to be formed. The peptide bond is formed by the transfer of the amino acid on the tRNA in the P site to the amino acid on the tRNA in the A site. The reaction joining two amino acids in a peptide bond is catalyzed by the rRNA of the large subunit of the ribosome. The cleavage of the high-energy bond linking the amino acid to the tRNA provides the energy for peptide bond formation between the carboxyl group of the first amino acid and the amino group of the second amino acid. Thereby, the initiating methionine retains its position as the first amino acid at the amino terminus of the protein. Fourth, the ribosome then slides along the mRNA so that the tRNAs are shifted in their positions in the ribosome. The tRNA in the P site, which is no longer carrying an amino acid, occupies the E site and exits the ribosome. This tRNA will be recycled, charged again by aminoacyl-tRNA synthetase, and used anew in translation. The tRNA with the growing peptide chain (previously in the A site) now occupies the P site, and the A site is empty and ready to accept another charged amino acid. The four steps of elongation will be repeated until the ribosome encounters a stop codon and the protein is complete. The direction of protein synthesis is from the amino end (methionine as the first amino acid) toward the carboxyl end. The growing peptide (linked to the tRNA in the P site) is always added to new incoming tRNAs. Although the process of initiation and elongation described here is for a single ribosome associated with the mRNA, most mRNAs are translated by polyribosomes(several ribosomes spaced evenly apart along the mRNAs that are synthesizing proteins). So, a mRNA is covered with ribosomes engaged in the various phases of translation.
Translation termination occurs when the ribosome encounters one of the three stop codons (UAG, UGA and UAA). In place of a charged tRNA at the A site, these codons recruit proteins called release factors. The binding of the release factors causes the growing polypeptide chain to be released from the tRNA and the two ribosomal subunits to dissociate from each other and the mRNA. The free polypeptide assumes its native conformation and, in many cases, is modified in a variety of ways, including cleavage, glycosylation (addition of sugars) and phosphorylation before it is active. Many proteins actually acquire their conformation as the protein is being synthesized. Some require the aid of chaperone proteins to ensure the correct conformation is achieved.
Cotranslational Insertion of Proteins into the ER
The synthesis of nuclear and cytoplasmic proteins occurs on ribosomes that are free in the cytoplasm. However, proteins that will eventually be associated with membranes (the endoplasmic reticulum, the Golgi complex and the plasma membrane) or secreted from the cell are synthesized on ribosomes that are bound to the endoplasmic reticulum, generating the rough ER (RER). The ribosomes in the RER are identical to the pool of free ribosomes, however, they have been directed to the ER via the sequence of the newly synthesized proteins. Membrane and secreted proteins have a distinct short sequence at the amino terminus referred to as the signal sequence. As the protein is synthesized and the signal sequence emerges, it is recognized and bound by the signal-recognition particle (SRP). The SRP will direct the ribosome, along with its partially synthesized protein, to the ER (see Figure 7). The cytoplasmic side of the ER contains receptors that specifically bind to the SRP, thus recruiting the SRP and the entire ribosome to the ER. After correct association with the ER, the partially synthesized protein is threaded across the ER membrane through the translocation complex, which creates a channel for the protein to pass through. The SRP bound to the signal sequence is anchored to the translocation complex and the remainder of the protein will pass into the lumen of the ER as translation elongation proceeds. In most cases the signal sequence is cleaved and the protein is released into the lumen of the ER, where it eventually makes its way to the correct membrane (see the tutorial on Intracellular Compartments: ER, Golgi Complex).
Translation is the process of converting the nucleotide code of mRNA into the amino acid code of proteins. The genetic code is a triplet of nucleotides, referred to as a codon, and each specify one of the 20 amino acids. Nucleotides in the genetic code do not overlap, but the code is redundant; several codons can specify the same amino acid. In addition, the start codon specifies the amino acid methionine, as well as the initiation of translation, and three stop codons specify translation termination. The mRNA is translated in the 5' to 3' direction and has three potential reading frames, but only one will encode the correct protein. The tRNAs are the molecules that interpret the genetic code. Each tRNA has an anticodon, a region complementary to one codon. The appropriate amino acid is covalently linked to the tRNA by an animoacyl-tRNA synthetase, with one enzyme specific for each amino acid. The charged tRNA will anneal to the complementary codon and deliver the correct amino acid. The process of translation occurs on the ribosome, a large protein-RNA complex composed of a large and a small subunit. There are three distinct ribosomal binding sites for the tRNAs (the A, P and E sites). The incoming charged tRNA binds to the A site, the tRNA carrying the growing peptide binds to the P site, and the exiting tRNA binds to the E site. Although there are many proteins in the ribosome, it is the rRNAs that function in the binding of mRNAs and tRNAs, and in catalyzing the peptide bond formed between amino acids delivered by the tRNAs. Translation has three phases: initiation, elongation and termination. Initiation occurs when the small ribosomal subunit binds the mRNA at the 5' end; the ribosomal subunit is already loaded with the initiator tRNA (complementary to the start codon) in the P site. The small subunit scans the mRNA until the first start codon is encountered, and then the large subunit is recruited. Elongation proceeds by the recruitment of a tRNA (escorted by elongation factors) to the empty A site of the ribosome. If the tRNA is correct, it will anneal stably to the codon of the mRNA and be retained long enough for a peptide bond to form. The amino acid of the tRNA in the P site is cleaved and then linked to the amino acid of the tRNA in the A site. The ribosome moves along the mRNA so that the empty tRNA now occupies the E site, the tRNA with the growing peptide chain occupies the P site, and the A site is empty, ready again to accept another incoming tRNA. Elongation continues until the ribosome encounters a stop codon, which recruits release factors that signal the release of the peptide from the tRNA and the dissociation of the ribosome. Membrane proteins are inserted into the ER membrane as they are being translated. The first few amino acids of a membrane protein include a short signal sequence that will target the peptide and associated ribosome to the ER membrane. The signal recognition particle (SRP) binds the signal sequence and is, in turn, recognized and bound by the SRP receptors on the ER membrane. Once this is delivered to the membrane, the signal sequence is threaded through the membrane via the translocation complex. As the remainder of the protein is made, it is also threaded across the translocation complex. The signal sequence is cleaved and the protein is now located in the ER lumen. For integral membrane proteins, the protein is inserted into the ER membrane in a similar fashion, but when the stop-transfer signal in the protein is encountered, translocation across the ER membrane ceases, resulting in the protein being embedded in the ER membrane. Proteins that are synthesized on ribosomes associated with the ER membrane will eventually be transported to their designated membrane.