The Protein Synthesis Machinery
Protein synthesis, or translation of mRNA into protein, occurs with the help of ribosomes, tRNAs, and aminoacyl tRNA synthetases.
Explain the role played by ribosomes, tRNA, and aminoacyl tRNA synthetases in protein synthesis
- Ribosomes, macromolecular structures composed of rRNA and polypeptide chains, are formed of two subunits (in bacteria and archaea, 30S and 50S; in eukaryotes, 40S and 60S), that bring together mRNA and tRNAs to catalyze protein synthesis.
- Fully assembled ribosomes have three tRNA binding sites: an A site for incoming aminoacyl-tRNAs, a P site for peptidyl-tRNAs, and an E site where empty tRNAs exit.
- tRNAs (transfer ribonucleic acids), which serve to deliver the appropriate amino acid to the growing peptide chain, consist of a modified RNA chain with the appropriate amino acid covalently attached.
- tRNAs have a loop of unbasepaired nucleotides at one end of the molecule that contains three nucleotides that act as the anticodon that basepairs to the mRNA codon.
- Aminoacyl tRNA synthetases are enzymes that load the individual amino acids onto the tRNAs.
- ribosome: protein/mRNA complexes found in all cells that are involved in the production of proteins by translating messenger RNA
The Protein Synthesis Machinery
In addition to the mRNA template, many molecules and macromolecules contribute to the process of translation. The composition of each component may vary across species. For instance, ribosomes may consist of different numbers of rRNAs and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to archaea to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors.
A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the synthesis and assembly of rRNAs occurs in the nucleolus.
Ribosomes exist in the cytoplasm in prokaryotes and in the cytoplasm and on rough endoplasmic reticulum membranes in eukaryotes. Mitochondria and chloroplasts also have their own ribosomes, and these look more similar to prokaryotic ribosomes (and have similar drug sensitivities) than the cytoplasmic ribosomes. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation.E. coli have a 30S small subunit and a 50S large subunit, for a total of 70S when assembled (recall that Svedberg units are not additive). Mammalian ribosomes have a small 40S subunit and a large 60S subunit, for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs.
In bacteria, archaea, and eukaryotes, the intact ribosome has three binding sites that accomodate tRNAs: The A site, the P site, and the E site. Incoming aminoacy-tRNAs (a tRNA with an amino acid covalently attached is called an aminoacyl-tRNA) enter the ribosome at the A site. The peptidyl-tRNA carrying the growing polypeptide chain is held in the P site. The E site holds empty tRNAs just before they exit the ribosome.
Each mRNA molecule is simultaneously translated by many ribosomes, all reading the mRNA from 5′ to 3′ and synthesizing the polypeptide from the N terminus to the C terminus. The complete mRNA/poly-ribosome structure is called a polysome.
tRNAs in eukaryotes
The tRNA molecules are transcribed by RNA polymerase III. Depending on the species, 40 to 60 types of tRNAs exist in the cytoplasm. Specific tRNAs bind to codons on the mRNA template and add the corresponding amino acid to the polypeptide chain. (More accurately, the growing polypeptide chain is added to each new amino acid bound in by a tRNA.)
The transfer RNAs (tRNAs) are structural RNA molecules. In eukaryotes, tRNA mole are transcribed from tRNA genes by RNA polymerase III. Depending on the species, 40 to 60 types of tRNAs exist in the cytoplasm. Serving as adaptors, specific tRNAs bind to sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain. (More accurately, the growing polypeptide chain is added to each new amino acid brought in by a tRNA.) Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins.
Of the 64 possible mRNA codons (triplet combinations of A, U, G, and C) three specify the termination of protein synthesis and 61 specify the addition of amino acids to the polypeptide chain. Of the three termination codons, one (UGA) can also be used to encode the 21st amino acid, selenocysteine, but only if the mRNA contains a specific sequence of nucleotides known as a SECIS sequence. Of the 61 non-termination codons, one codon (AUG) also encodes the initiation of translation.
Each tRNA polynucleotide chain folds up so that some internal sections basepair with other internal sections. If just diagrammed in two dimensions, the regions where basepairing occurs are called stems, and the regions where no basepairs form are called loops, and the entire pattern of stems and loops that forms for a tRNA is called the “cloverleaf” structure. All tRNAs fold into very similar cloverleaf structures of four major stems and three major loops.
If viewed as a three-dimensional structure, all the basepaired regions of the tRNA are helical, and the tRNA folds into a L-shaped structure.
Each tRNA has a sequence of three nucleotides located in a loop at one end of the molecule that can basepair with an mRNA codon. This is called the tRNA’s anticodon. Each different tRNA has a different anticodon. When the tRNA anticodon basepairs with one of the mRNA codons, the tRNA will add an amino acid to a growing polypeptide chain or terminate translation, according to the genetic code. For instance, if the sequence CUA occurred on a mRNA template in the proper reading frame, it would bind a tRNA with an anticodon expressing the complementary sequence, GAU. The tRNA with this anticodon would be linked to the amino acid leucine.
Aminoacyl tRNA Synthetases
The process of pre-tRNA synthesis by RNA polymerase III only creates the RNA portion of the adaptor molecule. The corresponding amino acid must be added later, once the tRNA is processed and exported to the cytoplasm. Through the process of tRNA “charging,” each tRNA molecule is linked to its correct amino acid by a group of enzymes called aminoacyl tRNA synthetases. When an amino acid is covalently linked to a tRNA, the resulting complex is known as an aminoacyl-tRNA. At least one type of aminoacyl tRNA synthetase exists for each of the 21 amino acids; the exact number of aminoacyl tRNA synthetases varies by species. These enzymes first bind and hydrolyze ATP to catalyze the formation of a covalent bond between an amino acid and adenosine monophosphate (AMP); a pyrophosphate molecule is expelled in this reaction. This is called “activating” the amino acid. The same enzyme then catalyzes the attachment of the activated amino acid to the tRNA and the simultaneous release of AMP. After the correct amino acid covalently attached to the tRNA, it is released by the enzyme. The tRNA is said to be charged with its cognate amino acid. (the amino acid specified by its anticodon is a tRNA’s cognate amino acid.)
The Mechanism of Protein Synthesis
Protein synthesis involves building a peptide chain using tRNAs to add amino acids and mRNA as a blueprint for the specific sequence.
Describe the process of translation
- Protein synthesis, or translation, begins with a process known as pre-initiation, when the small ribosmal subunit, the mRNA template, initiator factors, and a special initiator tRNA, come together.
- During translocation and elongation, the ribosome moves one codon 3′ down the mRNA, brings in a charged tRNA to the A site, transfers the growing polypeptide chain from the P-site tRNA to the carboxyl group of the A-site amino acid, and ejects the uncharged tRNA at the E site.
- When a stop or nonsense codon (UAA, UAG, or UGA) is reached on the mRNA, the ribosome terminates translation.
- translation: a process occurring in the ribosome in which a strand of messenger RNA (mRNA) guides assembly of a sequence of amino acids to make a protein
The Mechanism of Protein Synthesis
As with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination.
Initiation of Translation
Protein synthesis begins with the formation of a pre-initiation complex. In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors (IFs; IF-1, IF-2, and IF-3), and a special initiator tRNA, called fMet-tRNA. The initiator tRNA basepairs to the start codon AUG (or rarely, GUG) and is covalently linked to a formylated methionine called fMet. Methionine is one of the 21 amino acids used in protein synthesis; formylated methionine is a methione to which a formyl group (a one-carbon aldehyde) has been covalently attached at the amino nitrogen. Formylated methionine is inserted by fMet-tRNA at the beginning of every polypeptide chain synthesized by E. coli, and is usually clipped off after translation is complete. When an in-frame AUG is encountered during translation elongation, a non-formylated methionine is inserted by a regular Met-tRNA. In E. coli mRNA, a sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (AGGAGG), interacts with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template.
In eukaryotes, a pre-initiation complex forms when an initiation factor called eIF2 ( eukaryotic initiation factor 2) binds GTP, and the GTP-eIF2 recruits the eukaryotic initiator tRNA to the 40s small ribosomal subunit. The initiator tRNA, called Met-tRNAi, carries unmodified methionine in eukaryotes, not fMet, but it is distinct from other cellular Met-tRNAs in that it can bind eIFs and it can bind at the ribosome P site. The eukaryotic pre-initiation complex then recognizes the 7-methylguanosine cap at the 5′ end of a mRNA. Several other eIFs, specifically eIF1, eIF3, and eIF4, act as cap-binding proteins and assist the recruitment of the pre-initiation complex to the 5′ cap. Poly (A)-Binding Protein (PAB) binds both the poly (A) tail of the mRNA and the complex of proteins at the cap and also assists in the process. Once at the cap, the pre-initiation complex tracks along the mRNA in the 5′ to 3′ direction, searching for the AUG start codon. Many, but not all, eukaryotic mRNAs are translated from the first AUG sequence. The nucleotides around the AUG indicate whether it is the correct start codon.
Once the appropriate AUG is identified, eIF2 hydrolyzes GTP to GDP and powers the delivery of the tRNAi-Met to the start codon, where the tRNAi anticodon basepairs to the AUG codon. After this, eIF2-GDP is released from the complex, and eIF5-GTP binds. The 60S ribosomal subunit is recruited to the pre-initiation complex by eIF5-GTP, which hydrolyzes its GTP to GDP to power the assembly of the full ribosome at the translation start site with the Met-tRNAi positioned in the ribosome P site. The remaining eIFs dissociate from the ribosome and translation is ready to begins.
In archaea, translation initiation is similar to that seen in eukaryotes, except that the initiation factors involved are called aIFs (archaeal inititiaion factors), not eIFs.
The basics of elongation are the same in prokaryotes and eukaryotes. The intact ribosome has three compartments: the A site binds incoming aminoacyl tRNAs; the P site binds tRNAs carrying the growing polypeptide chain; the E site releases dissociated tRNAs so that they can be recharged with amino acids. The initiator tRNA, rMet-tRNA in E. coli and Met-tRNAi in eukaryotes and archaea, binds directly to the P site. This creates an initiation complex with a free A site ready to accept the aminoacyl-tRNA corresponding to the first codon after the AUG.
The aminoacyl-tRNA with an anticodon complementary to the A site codon lands in the A site. A peptide bond is formed between the amino group of the A site amino acid and the carboxyl group of the most-recently attached amino acid in the growing polypeptide chain attached to the P-site tRNA.The formation of the peptide bond is catalyzed by peptidyl transferase, an RNA-based enzyme that is integrated into the large ribosomal subunit. The energy for the peptide bond formation is derived from GTP hydrolysis, which is catalyzed by a separate elongation factor.
Catalyzing the formation of a peptide bond removes the bond holding the growing polypeptide chain to the P-site tRNA. The growing polypeptide chain is transferred to the amino end of the incoming amino acid, and the A-site tRNA temporarily holds the growing polypeptide chain, while the P-site tRNA is now empty or uncharged.
The ribosome moves three nucleotides down the mRNA. The tRNAs are basepaired to a codon on the mRNA, so as the ribosome moves over the mRNA, the tRNAs stay in place while the ribosome moves and each tRNA is moved into the next tRNA binding site. The E site moves over the former P-site tRNA, now empty or uncharged, the P site moves over the former A-site tRNA, now carrying the growing polypeptide chain, and the A site moves over a new codon. In the E site, the uncharged tRNA detaches from its anticodon and is expelled. A new aminoacyl-tRNA with an anticodon complementary to the new A-site codon enters the ribosome at the A site and the elongation process repeats itself. The energy for each step of the ribosome is donated by an elongation factor that hydrolyzes GTP.
Termination of translation occurs when the ribosome moves over a stop codon (UAA, UAG, or UGA). There are no tRNAs with anticodons complementary to stop codons, so no tRNAs enter the A site. Instead, in both prokaryotes and eukaryotes, a protein called a release factor enters the A site. The release factors cause the ribosome peptidyl transferase to add a water molecule to the carboxyl end of the most recently added amino acid in the growing polypeptide chain attached to the P-site tRNA. This causes the polypeptide chain to detach from its tRNA, and the newly-made polypeptide is released. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.
Protein Folding, Modification, and Targeting
In order to function, proteins must fold into the correct three-dimensional shape, and be targeted to the correct part of the cell.
Discuss how post-translational events affect the proper function of a protein
- Protein folding is a process in which a linear chain of amino acids attains a defined three-dimensional structure, but there is a possibility of forming misfolded or denatured proteins, which are often inactive.
- Proteins must also be located in the correct part of the cell in order to function correctly; therefore, a signal sequence is often attached to direct the protein to its proper location, which is removed after it attains its location.
- Protein misfolding is the cause of numerous diseases, such as mad cow disease, Creutzfeldt-Jakob disease, and cystic fibrosis.
- prion: a self-propagating misfolded conformer of a protein that is responsible for a number of diseases that affect the brain and other neural tissue
- chaperone: a protein that assists the non-covalent folding/unfolding of other proteins
After being translated from mRNA, all proteins start out on a ribosome as a linear sequence of amino acids. This linear sequence must “fold” during and after the synthesis so that the protein can acquire what is known as its native conformation. The native conformation of a protein is a stable three-dimensional structure that strongly determines a protein’s biological function. When a protein loses its biological function as a result of a loss of three-dimensional structure, we say that the protein has undergone denaturation. Proteins can be denatured not only by heat, but also by extremes of pH; these two conditions affect the weak interactions and the hydrogen bonds that are responsible for a protein’s three-dimensional structure. Even if a protein is properly specified by its corresponding mRNA, it could take on a completely dysfunctional shape if abnormal temperature or pH conditions prevent it from folding correctly. The denatured state of the protein does not equate with the unfolding of the protein and randomization of conformation. Actually, denatured proteins exist in a set of partially-folded states that are currently poorly understood. Many proteins fold spontaneously, but some proteins require helper molecules, called chaperones, to prevent them from aggregating during the complicated process of folding.
Protein Modification and Targeting
During and after translation, individual amino acids may be chemically modified and signal sequences may be appended to the protein. A signal sequence is a short tail of amino acids that directs a protein to a specific cellular compartment. These sequences at the amino end or the carboxyl end of the protein can be thought of as the protein’s “train ticket” to its ultimate destination. Other cellular factors recognize each signal sequence and help transport the protein from the cytoplasm to its correct compartment. For instance, a specific sequence at the amino terminus will direct a protein to the mitochondria or chloroplasts (in plants). Once the protein reaches its cellular destination, the signal sequence is usually clipped off.
It is very important for proteins to achieve their native conformation since failure to do so may lead to serious problems in the accomplishment of its biological function. Defects in protein folding may be the molecular cause of a range of human genetic disorders. For example, cystic fibrosis is caused by defects in a membrane-bound protein called cystic fibrosis transmembrane conductance regulator (CFTR). This protein serves as a channel for chloride ions. The most common cystic fibrosis-causing mutation is the deletion of a Phe residue at position 508 in CFTR, which causes improper folding of the protein. Many of the disease-related mutations in collagen also cause defective folding.
A misfolded protein, known as prion, appears to be the agent of a number of rare degenerative brain diseases in mammals, like the mad cow disease. Related diseases include kuru and Creutzfeldt-Jakob. The diseases are sometimes referred to as spongiform encephalopathies, so named because the brain becomes riddled with holes. Prion, the misfolded protein, is a normal constituent of brain tissue in all mammals, but its function is not yet known. Prions cannot reproduce independently and not considered living microoganisms. A complete understanding of prion diseases awaits new information about how prion protein affects brain function, as well as more detailed structural information about the protein. Therefore, improved understanding of protein folding may lead to new therapies for cystic fibrosis, Creutzfeldt-Jakob, and many other diseases.