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Some very basic parts of transcription/translation seem to be left out in various literature. I can't find the answer to this anywhere:
How exactly is tRNA synthesized? I realize that mRNA is synthesized through transcription and I know a lot about that. However tRNA is supposedly synthesized the same way but every time you read about transcription they just talk about how the mRNA then gets this and that… ?
Where do the amino acids get attached? Is it in the nucleus or outside the nucleus?
A pre-tRNA is transcribed from tRNA genes in DNA by RNA polymerase III. Processing occurs in the nucleus, where a 5' sequence is cleaved by RNase P, the 3's CCA motif is added, and ~10% of the nucleotides are substituted. The tRNA are transported out via the pore complexes. Aminoacyl-tRNA synthetase enzymes attach amino acids in the cytoplasm in a 2-step reaction that requires ATP. You'll find there's a unique splicing mechanism in tRNA that additionally splices out an anticodon intron which is abesnt in mature tRNA's:
The wikipedia article notes RNA Pol III generally recognizes internal control elements rather than upstream control elements as in a normal gene.
Source: Molecular Cell Biology. 4th edition.
Addendum: I said in my post that tRNA is charged in the cytoplasm, this is somewhat true. In mammalian cells, we also see that tRNA are charged in the nucleus as well, and it might aid in the export of some of these charged tRNAs. (Source)
Role of Ribosomes in Protein Synthesis (With Diagram)
Ribosomes provide framework on which protein synthesis takes place. The mRNA binds to the 30S subunit of ribosome to form initiation complex. The main role of ribosome is its ability to catalyse the formation of peptide bonds between amino acids, so that the amino acids are incorporated into proteins.
Ribosomes are dense granules without covering membranes. They were first observed by Palade. Bacterial ribosomes contain 65% RNA (rRNA) and 35% proteins. They have a diameter of 18 nm. Ribosomes are found in all cells. E. coli has 10,000 ribosomes, and form about 25% of total mass of bacterial cell. A mammalian cell contains about 10 million ribosomes.
A Ribosome has Two Subunits:
Each ribosome has two unequal subunits, a large and a small subunit. Bacterial ribosomes are composed of two subunits of 30S and 50S sedimentation coefficient in sucrose. They have combined sedimentation coefficient of 70S. Both subunits contain many proteins and at least one large rRNA.
Eukaryotic ribosomes are larger than bacterial ribosomes. They have two unequal subunits of 40S and 60S having a combined sedimentation coefficient of 80S. Ribosomes are measured in terms of their rate of sedimentation measured in Svedberg units (S). IS = 10
13 second. All ribosomes in a given cell are identical. Components of ribosome can separate and can reassemble spontaneously. A ribosome has a core of rRNA the proteins attached on the surface.
In prokaryotes both 30S and 50S subunits have rRNA and protein molecule components.
Similarly eukaryotic ribosome ribosome have the following components:
40S subunit: 18S molecule + 30 proteins
60S subunit: 5S, 5.8S and rRNA +50 proteins.
The 70S ribosome structure is not symmetrical. 70S ribosome is divided into four regions. These are head, neck, body and platform. The 50S subunit has a central protuberance having 5S rRNA and stalk having proteins.
Most of the proteins are basic proteins and have strong association with RNA, which is acidic in nature. Ribosomal RNA (rRNA) represents more than 80% of the total RNA present in the bacterial cell.
Two subunits of ribosome associate and dissociate depending upon the concentration of magnesium. About 70% of ribosomal RNA is double stranded and helical with various stems and loops due to base pairing between complementary regions. The interaction of 16S rRNA and mRNA helps the 30S subunit to recognise the starting end of mRNA. The ribosome binding site in prokaryotes lies near the 5′-end of mRNA upstream of start codon AUG.
Between 5′-end of mRNA and AUG codon there are many bases. Out of these there is a sequence of 5′-AGGAGGU-3′. This is called Shine-Dalgarno sequence and lies 4-7 bases upstream of AUG. The 3′-end region of 16S rRNA has a complementary sequence of 3′- AUUCCUCCA-5′. This sequence binds mRNA to ribosome.
A ribosome has two channels in it. The linear mRNA enters and escapes through one channel which has the decoding centre. This channel is accessible to the charged tRNAs. The newly synthesized polypeptide chain escapes through the other channel.
Small subunit of ribosome contains the decoding centre in which charged tRNAs decode the codons of mRNA. Large subunit contains peptidyl transferase center, which forms peptide bonds between successive amino acids. The mRNA binds to the 3′-end of 16S RNA in 30S subunit of ribosome. The 30S subunit, mRNA and charged tRNA combine to form pre- initiation complex along-with initiation factors and GTP. Later 50S subunit of ribosome joins to form 70S initiation complex.
The main role of ribosome is the formation of peptide bonds between successive amino acids of the newly synthesized peptide chain.
There are two tRNA binding sites on ribosome. The first site is called ‘P’ site or peptidyl site. The second site is called ‘A’ site or amino acyl site. Only the initiator tRNA enters the ‘P’ site. All other tRNAs enter the ‘A’ site.
The main role of ribosome is the formation of peptide bonds between successive amino acids. The peptide bond is formed between amino acid at “A” site and peptide chain at “P” thus lengthening the chain by one amino acid. It was discovered that the peptidyltransferase which catalizes the peptide bond formation between successive amino acids consists of several proteins and a 23S rRNA molecule. This 23S rRNA is a ribozyme and is responsible for catalyzing peptide bond formation between successive amino acids.
In addition to these two sites ‘P’ and ‘A’ site, a third site ‘E’ or exit site is present. Deacylated tRNA (deprived of amino acid) moves from ‘P’ site to ‘E’ site from where it is ejected out.
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Transfer RNA (tRNA), small molecule in cells that carries amino acids to organelles called ribosomes, where they are linked into proteins. In addition to tRNA there are two other major types of RNA: messenger RNA (mRNA) and ribosomal RNA (rRNA). By 1960 the involvement of tRNAs in the assembly of proteins was demonstrated by several scientists, including American biochemist Robert William Holley, who also developed techniques to separate different transfer RNAs from cells and determined the composition of the tRNA that incorporates the amino acid alanine into protein molecules.
Ribosomal molecules of mRNA determine the order of tRNA molecules that are bound to nucleotide triplets (codons). The order of tRNA molecules ultimately determines the amino acid sequence of a protein because molecules of tRNA catalyze the formation of peptide bonds between the amino acids, linking them together to form proteins. The newly formed proteins detach themselves from the ribosome site and migrate to other parts of the cell for use.
Molecules of tRNA typically contain fewer than 100 nucleotide units and fold into a characteristic cloverleaf structure. Specialized tRNAs exist for each of the 20 amino acids needed for protein synthesis, and in many cases more than one tRNA for each amino acid is present. The 61 codons used to code amino acids can be read by many fewer than 61 distinct tRNAs. In the bacterium Escherichia coli a total of 40 different tRNAs are used to translate the 61 codons. The amino acids are loaded onto the tRNAs by specialized enzymes called aminoacyl tRNA synthetases, usually with one synthetase for each amino acid. However, in some organisms, less than the full complement of 20 synthetases are required because some amino acids, such as glutamine and asparagine, can be synthesized on their respective tRNAs. All tRNAs adopt similar structures because they all have to interact with the same sites on the ribosome.
This article was most recently revised and updated by Kara Rogers, Senior Editor.
8.6: Protein synthesis: translation (RNA to polypeptide)
- Contributed by Michael W. Klymkowsky and Melanie M. Cooper
- Professors (MSCD and Chemistry) at University of Colorado Boulder and Michigan State University
Translation involves a complex cellular organelle, the ribosome, which together with a number of accessory factors reads the code in a mRNA molecule and produces the appropriate polypeptide 235 . The ribosome is the site of polypeptide synthesis. It holds the various components (the mRNA, tRNAs, and accessory factors) in appropriate juxtaposition to one another to catalyze polypeptide synthesis. But perhaps we are getting ahead of ourselves. For one, what exactly is a tRNA?
The process of transcription is also used to generate other types of RNAs these play structural, catalytic, and regulatory roles within the cell. Of these non-mRNAs, two are particularly important in the context of polypeptide synthesis. The first are molecules known as transfer RNAs (tRNAs). These small single stranded RNA molecules fold back on themselves to generate a compact L-shaped structure. In the bacterium E. coli, there are 87 genes that encode tRNAs (there are over 400 such tRNA encoding genes in human). For each amino acid and each codon there are one or more tRNAs. The only exception being the stop codons, for which there are no tRNAs. A tRNA specific for the amino acid phenylalanine would be written tRNA Phe . Two parts of the tRNA molecule are particularly important and functionally linked: the part that recognizes the codon on the mRNA (in the mRNA-ribosome complex) and the amino acid acceptor stem, which is where an amino acid is attached to the tRNA. Each specific type of tRNA can recognize a particular codon in an mRNA through base pairing interactions with what is known as the anti-codon. The rest of the tRNA molecule mediates interactions with protein catalysts (enzymes) known as amino acyl tRNA synthetases. There is a distinct amino acyl tRNA synthetase for each amino acid: there is a phenylalanine-tRNA synthetase and a proline-tRNA synthetase, etc. An amino acyl tRNA synthetase binds the appropriate tRNA and the appropriate amino acid and, through a reaction coupled to a thermodynamically favorable nucleotide triphosphate hydrolysis reaction, catalyzes the formation of a covalent bond between the amino acid acceptor stem of the tRNA and the amino acid, to form what is known as a charged or amino acyl-tRNA. The loop containing the anti-codon is located at the other end of the tRNA molecule. As we will see, in the course of polypeptide synthesis, the amino acid group attached to the tRNA&rsquos acceptor stem will be transferred from the tRNA to the growing polypeptide.
Ribosomes: Ribosomes are composed of roughly equal amounts (by mass) of ribosomal (rRNAs) and ribosomal polypeptides. An active ribosome is composed of a small and a large ribosomal subunit. In the bacterium E. coli, the small subunit is composed of 21 different polypeptides and a 1542 nucleotide long rRNA molecule, while the large subunit is composed of 33 different polypeptides and two rRNAs, one 121 nucleotides long and the other 2904 nucleotides long 236 . It goes without saying (so why are we saying it?) that each ribosomal polypeptide and RNA is itself a gene product. The complete ribosome has a molecular weight of
3 x 10 6 daltons. One of the rRNAs is an evolutionarily conserved catalyst, known as a ribozyme (in contrast to protein based catalysts, which are known as enzymes). This rRNA lies at the heart of the ribosome and catalyzes the transfer of an amino acid bound to a tRNA to the carboxylic acid end of the growing polypeptide chain. RNA based catalysis is a conserved feature of polypeptide synthesis and appears to represent an evolutionarily homologous trait.
The growing polypeptide chain is bound to a tRNA, known as the peptidyl tRNA. When a new aa-tRNA enters the ribosome&rsquos active site (site A), the growing polypeptide is added to it, so that it becomes the peptidyl tRNA (with a newly added amino acid, the amino acid originally associated with incoming aa-tRNA). This attached polypeptide group is now one amino acid longer.
The cytoplasm of cells is packed with ribosomes. In a rapidly growing bacterial cell,
25% of the total cell mass is ribosomes. Although structurally similar, there are characteristic differences between the ribosomes of bacteria, archaea, and eukaryotes. This is important from a practical perspective. For example, a number of antibiotics selectively inhibit polypeptide synthesis by bacterial, but not eukaryotic ribosomes. Both chloroplasts and mitochondria have ribosomes of the bacterial type. This is another piece of evidence that chloroplasts and mitochondria are descended from bacterial endosymbionts and a reason that translational blocking anti-bacterial antibiotics are mostly benign, since most of the ribosomes inside a eukaryotic cell are not effected by them.
14.E: Translation - Protein synthesis (Exercises)
- Contributed by Ross Hardison
- T. Ming Chu Professor (Biochemistry and Molecular Biology) at The Pennsylvania State University
14.1 (POB) Methionine Has Only One Codon.
Methionine is one of the two amino acids having only one codon. Yet the single codon for methionine can specify both the initiating residue and interior Met residues of polypeptides synthesized by E. coli. Explain exactly how this is possible.
14.2 Are the following statements concerning aminoacyl‑tRNA synthetase true or false?
a) Two distinct classes of the enzymes have been defined that are not very related to each other.
b) The enzymes scan previously‑synthesized aminoacyl‑tRNAs and cleave off any amino acids that are linked to the incorrect tRNA.
c) Proofreading can occur at the formation of either the aminoacyl‑adenylate intermediate (in some synthetases) or at the aminoacyl‑tRNA (in other synthetases) to insure that the correct amino acid is attached to a given tRNA.
d) The product of the reaction has a high‑energy ester bond between the carboxyl of an amino acid and a hydroxyl on the terminal ribose of the tRNA.
14.3 A preparation of ribosomes in the process of synthesizing the polypeptide insulin was incubated in the presence of all 20 radiolabeled amino acids, tRNA's, aminoacyl-tRNA synthetases and other components required for protein synthesis. All the amino acids have the same specific radioactivity (counts per minute per nanomole of amino acid). It takes ten minutes to synthesize a complete insulin chain (from initiation to termination) in this system. After incubation for 1 minute, the completed insulin chains were cleaved with trypsin and the radioactivity of the fragments determined.
a) Which tryptic fragment has the highest specific activity?
b) In the same system described above, the insulin polypeptide chains still attached to the ribosomes after ten minutes were isolated, cleaved with trypsin, and the specific activity of each tryptic peptide determined. Which peptide has the highest specific activity?
14.4 Which component of the protein synthesis machinery of E. colicarries out the function listed for each statement.
a) Translocation of the peptidyl-tRNA from the A site to the P site of the ribosome.
b) Binding of f-Met-tRNA to the mRNA on the small ribosomal subunit.
c) Recognition of the termination codons UAG and UAA.
d) Holds the initiator AUG in register for formation of the initiation complex (via base pairing).
14.5 a) In the initiation of translation in E. coli, which ribosomal subunit does the mRNA initially bind to?
b) What nucleotide sequences in the mRNA are required to direct the mRNA to the initial binding site on the ribosome?
c) What other factors are required to form an initiation complex?
14.6 What steps in the activation of amino acids and elongation of a polypeptide chain require hydrolysis of high energy phosphate bonds? What enzymes catalyze these steps or which protein factors are required?
14.7(POB) Maintaining the Fidelity of Protein Synthesis
The chemical mechanisms used to avoid errors in protein synthesis are different from those used during DNA replication. DNA polymerases utilize a 3' ® 5' exonuclease proofreading activity to remove mispaired nucleotides incorrectly inserted into a growing DNA strand. There is no analogous proofreading function on ribosomes and, in fact, the identity of amino acids attached to incoming tRNAs and added to the growing polypeptide is never checked. A proofreading step that hydrolyzed the last peptide bond formed when an incorrect amino acid was inserted into a growing polypeptide (analogous to the proofreading step of DNA polymerases) would actually be chemically impractical. Why? (Hint: Consider how the link between the growing polypeptide and the mRNA is maintained during the elongation phase of protein synthesis.)
14.8 (POB) Expressing a Cloned Gene.
You have isolated a plant gene that encodes a protein in which you are interested. What are the sequences or sites that you will need to get this gene transcribed, translated, and regulated in E. coli.)?
14.9 The three codons AUU, AUC, and AUA encode isoleucine. They correspond to "hybrid" between a codon family (4 codons) and a codon pair (2 codons). The single codon AUG encodes methionine. Given the prevalence of codon pairs and families for other amino acids, what are hypotheses for how this situation for isoleucine and methionine could have evolved?
14.10 Use the following processes to answer parts a-c:
- synthesis of aminoacyl-tRNA from an amino acid and tRNA.
- binding of aminoacyl-tRNA to the ribosome for elongation.
- formation of the peptide bond between peptidyl-tRNA and aminoacyl-tRNA on the ribosome.
- translocation of peptidyl-tRNA from the A site to the P site on the ribosome.
- assembly of a spliceosome for removal of introns from nuclear pre-mRNA.
- removal of introns from nuclear pre-mRNA after assembly of a spliceosome.
- synthesis of a 5' cap on eukaryotic mRNA.
(a) Which of the above processes require ATP?
(b) Which of the above processes require GTP?
(c) For which of the above processes is there evidence that RNA is used as a catalyst?
The idea that DNA makes RNA and RNA makes protein is sometimes referred to as the central dogma of molecular biology the term was coined by Francis Crick, one of the co-discoverers of DNA's structure.
1. From DNA to RNA: How does the information in the DNA molecule get turned into the proteins that give our bodies structure? We won't go into this in great detail but, to give a big picture overview, here's what happens: DNA, which is a double stranded molecule, unzips down the center through the action of an enzyme called RNA polymerase. An RNA template is laid down on top of one of the DNA strands, making a near mirror image (instead of Thymine, RNA uses Uracil) of the DNA code, as shown in the table below. This template is laid down in the 5' to 3' direction. (Remember that the strands are anti-parallel.) The RNA strand is now called messenger RNA, or mRNA, and leaves the nucleus of the cell. This process of converting DNA information into RNA information is called transcription.
|Whenever the DNA had this nucleotide base.||the RNA will have this nucleotide base|
|Cytosine (C)||Guanine (G)|
|Guanine (G)||Cytosine (C)|
|Adenine (A)||Uracil (U)|
|Thymine (T)||Adenine (A)|
2. From RNA to Amino Acid: The mRNA arrives at a ribosome. Each three letter snippet of RNA is called a codon. The RNA codons are read by the ribosome. Each codon calls for the synthesis of a particular amino acid. There are about 20 biologically important amino acids. Read the table below from left to right to look up the amino acid that is called for by each codon. For example, if the RNA codon reads "CAG," the amino acid that will be synthesized is Glutamine. If the RNA codon reads "ACG," the amino acid that will be synthesized is Threonine. (Note that, in some cases, there is more than one codon that will result in the same amino acid.) As the ribosome moves across the mRNA strand, reading the 3-letter codons, a special transport molecule called transfer RNA (tRNA) is dipatched to pick up the amino acid specified by the codon. The tRNA delivers the correct amino acid back to the ribosome, lands on the mRNA codon (the tRNA molecule has a corresponding anti-codon), and releases its amino acid cargo, where it is attached to a growing peptide chain. This process of converting RNA information into amino acids is called translation.
|2nd base||3rd |
|U||UUU||(Phe/F) Phenylalanine||UCU||(Ser/S) Serine||UAU||(Tyr/Y) Tyrosine||UGU||(Cys/C) Cysteine||U|
|UUA||(Leu/L) Leucine||UCA||UAA||Stop (Ochre)||UGA||Stop (Opal)||A|
|UUG||UCG||UAG||Stop (Amber)||UGG||(Trp/W) Tryptophan||G|
|C||CUU||CCU||(Pro/P) Proline||CAU||(His/H) Histidine||CGU||(Arg/R) Arginine||U|
|A||AUU||(Ile/I) Isoleucine||ACU||(Thr/T) Threonine||AAU||(Asn/N) Asparagine||AGU||(Ser/S) Serine||U|
|AUA||ACA||AAA||(Lys/K) Lysine||AGA||(Arg/R) Arginine||A|
|G||GUU||(Val/V) Valine||GCU||(Ala/A) Alanine||GAU||(Asp/D) Aspartic acid||GGU||(Gly/G) Glycine||U|
|GUA||GCA||GAA||(Glu/E) Glutamic acid||GGA||A|
3. From Amino Acid to Protein: After each new amino acid comes off the assembly line, it is joined to the end of the growing peptide chain. Remember that while the base unit of each amino acid is identical, the attached R group is what gives each amino acid its unique physical properties. A good way to imagine this is to think of a chain composed of beads of different types.
Here's an animation that shows you how the whole process works.
3-D structure: Imagine that each chain of amino acids, the peptide, is like a long piece of yarn. The "primary structure" of a protein is the makeup of the strands themselves. The "secondary structure" of a protein is the way in which these strands are woven together, as strands of hair are woven together into a braid, or as fibers are interlaced into a sheet of fabric. The "tertiary structure" of a protein is the way in which those braided strands are twisted and folded to make a complex three-dimensional structure. For example, think about how yarn can be knit into a hat, sock, or mitten each with a unique 3-D shape.
Mutation: Now that you understand how to read DNA, let's think about what happens when a mutation occurs. A mutation is a change to the DNA. Changes can happen when a copying error occurs or when the DNA is damaged by radiation or chemical toxins. When changes occur, it's usually a bad thing but, depending on the kind of change, it can be minor or very serious. Here are some possibilities: A single letter in the DNA sequence can be substituted by another. A letter (or sequence of letters) can be inserted or deleted. A sequence of letters can be inverted (cut out, flipped and re-inserted). A sequence of letters can be duplicated one or more times. Imagine that instead of the Cs, Gs, As and Ts of the DNA, we use a sentence where each word is a codon. Here's what that sentence might look like after each kind of mutation:
Original: The quick brown fox jumped over the lazy dog.
Substitution: The quick brown fog jumped over the lazy dog. (The x became a g. The sentence remains intelligible but meaning has shifted slightly. This is also sometimes called a "point mutation" because it affects only a single point on the DNA sequence.)
Insertion: The quick brown fox gjumpe dove rth elaz ydog. (A single letter g inserted after the x shifts letters to the right, changing the breaks between our word codons and making everything after the insertion nonsensical. However, we accidentally created the word "dove" which is was unintentional but does have meaning. Maybe this is a beneficial mutation?
Deletion: The quick brown foj umpedo vert hel azyd og. (After the single letter x is deleted, every letter is shifted one position to the left. The sentence makes sense up to the point of the mutation, but then becomes scrambled.)
Multi-letter deletion: The quick fox jumped over the lazy dog. (If an entire codon gets deleted, there is no shift to the left or right. The sentence remains intelligible but the meaning might change. If the deletion doesn't happen exactly at the word codon breaks however, this would end up more like the previous two examples.)
Inversion: The quick muj xof nworbped over the lazy dog. (The inverted section makes no sense but the rest of the words are unaffected.)
Duplication: The quick brown fox brown fox brown fox jumped over the lazy dog. (The duplication of brown fox makes the sentence a bit harder to figure out but, because full codons were duplicated, it remains intelligible. If the duplication happened across codons, it might be a much bigger mess.)
Translocation: The DNA fragment is cut from one chromosome and pasted to a different one.
The Mechanism of Protein Synthesis
Translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between bacterial and eukaryotic translation.
The initiation of protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator tRNA carrying N-formyl-methionine (fMet-tRNA fMet ) (Figure 4). The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet). Because of its involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli. In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome.
In eukaryotes, initiation complex formation is similar, with the following differences:
- The initiator tRNA is a different specialized tRNA carrying methionine, called Met-tRNAi
- Instead of binding to the mRNA at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 5′ cap of the eukaryotic mRNA, then tracks along the mRNA in the 5′ to 3′ direction until the AUG start codon is recognized. At this point, the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit.
Figure 4. Translation in bacteria begins with the formation of the initiation complex, which includes the small ribosomal subunit, the mRNA, the initiator tRNA carrying N-formyl-methionine, and initiation factors. Then the 50S subunit binds, forming an intact ribosome.
In prokaryotes and eukaryotes, the basics of elongation of translation are the same. In E. coli, the binding of the 50S ribosomal subunit to produce the intact ribosome forms three functionally important ribosomal sites: The A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one notable exception to this assembly line of tRNAs: During initiation complex formation, bacterial fMet−tRNA fMet or eukaryotic Met-tRNAi enters the P site directly without first entering the A site, providing a free A site ready to accept the tRNA corresponding to the first codon after the AUG.
Elongation proceeds with single-codon movements of the ribosome each called a translocation event. During each translocation event, the charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal. Ribosomal movements, or steps, are induced by conformational changes that advance the ribosome by three bases in the 3′ direction. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based ribozyme that is integrated into the 50S ribosomal subunit. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled. Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site and translocation, require energy derived from GTP hydrolysis, which is catalyzed by specific elongation factors. Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds.
The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered for which there is no complementary tRNA. On aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that result in the P-site amino acid detaching from its tRNA, releasing the newly made polypeptide. The small and large ribosomal subunits dissociate from the mRNA and from each other they are recruited almost immediately into another translation init iation complex.
In summary, there are several key features that distinguish prokaryotic gene expression from that seen in eukaryotes. These are illustrated in Figure 6 and listed in Table 1.
Figure 6. (a) In prokaryotes, the processes of transcription and translation occur simultaneously in the cytoplasm, allowing for a rapid cellular response to an environmental cue. (b) In eukaryotes, transcription is localized to the nucleus and translation is localized to the cytoplasm, separating these processes and necessitating RNA processing for stability.
30S (small subunit) with 16S rRNA subunit
40S (small subunit) with 18S rRNA subunit
Bovee et al. show that, at least in one case, aminoacyl-tRNA synthetases can tell the difference between the right and the wrong tRNA before they ever start catalysis. The authors also show that if the enzyme binds amino-acid-adenylate first, it is even more specific during tRNA binding. Previous kinetic data have also proven that aminoacyl-tRNA synthetases reject wrong tRNAs during catalysis, and Bovee et al. cite a recent model proposing that tRNA binding itself might happen in several steps - the tRNA might change conformation after its first contact with the enzyme. Specificity, therefore, seems to happen at multiple stages during the synthetase reaction.
Because amino-tRNA synthetase specificity is apparently so complicated, this paper may be probing what will prove to be a minor part of mechanism. What the field needs now is to find the rate-determining step in the synthetase reaction. That may tell us where the 'most important' specificity screen is, for both tRNA and amino-acid binding.
15.5 Ribosomes and Protein Synthesis
By the end of this section, you will be able to do the following:
- Describe the different steps in protein synthesis
- Discuss the role of ribosomes in protein synthesis
The synthesis of proteins consumes more of a cell’s energy than any other metabolic process. In turn, proteins account for more mass than any other component of living organisms (with the exception of water), and proteins perform virtually every function of a cell. The process of translation, or protein synthesis, involves the decoding of an mRNA message into a polypeptide product. Amino acids are covalently strung together by interlinking peptide bonds in lengths ranging from approximately 50 to more than 1000 amino acid residues. Each individual amino acid has an amino group (NH2) and a carboxyl (COOH) group. Polypeptides are formed when the amino group of one amino acid forms an amide (i.e., peptide) bond with the carboxyl group of another amino acid (Figure 15.15). This reaction is catalyzed by ribosomes and generates one water molecule.
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 example, 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 human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors. (Note: A ribosome can be thought of as an enzyme whose amino acid binding sites are specified by mRNA.)
Link to Learning
Click through the steps of this PBS interactive to see protein synthesis in action.
Even before an mRNA is translated, a cell must invest energy to build each of its ribosomes. In E. coli, there are between 10,000 and 70,000 ribosomes present in each cell at any given time. A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the nucleolus is completely specialized for the synthesis and assembly of rRNAs.
Ribosomes exist in the cytoplasm of prokaryotes and in the cytoplasm and rough endoplasmic reticulum of eukaryotes. Mitochondria and chloroplasts also have their own ribosomes in the matrix and stroma, which look more similar to prokaryotic ribosomes (and have similar drug sensitivities) than the ribosomes just outside their outer membranes in the cytoplasm. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation. In E. coli, the small subunit is described as 30S, and the large subunit is 50S, for a total of 70S (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. Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction: 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 .
The tRNAs are structural RNA molecules that were transcribed from genes by RNA polymerase III. Depending on the species, 40 to 60 types of tRNAs exist in the cytoplasm. Transfer RNAs serve as adaptor molecules. Each tRNA carries a specific amino acid and recognizes one or more of the mRNA codons that define the order of amino acids in a protein. Aminoacyl-tRNAs bind to the ribosome and add the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins.
Of the 64 possible mRNA codons—or 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 these 61, one codon (AUG) also encodes the initiation of translation. Each tRNA anticodon can base pair with one or more of the mRNA codons for its amino acid. For instance, if the sequence CUA occurred on an mRNA template in the proper reading frame, it would bind a leucine tRNA expressing the complementary sequence, GAU. The ability of some tRNAs to match more than one codon is what gives the genetic code its blocky structure.
As the adaptor molecules of translation, it is surprising that tRNAs can fit so much specificity into such a small package. Consider that tRNAs need to interact with three factors: 1) they must be recognized by the correct aminoacyl synthetase (see below) 2) they must be recognized by ribosomes and 3) they must bind to the correct sequence in mRNA.
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 one of a group of enzymes called aminoacyl tRNA synthetases . At least one type of aminoacyl tRNA synthetase exists for each of the 20 amino acids the exact number of aminoacyl tRNA synthetases varies by species. These enzymes first bind and hydrolyze ATP to catalyze a high-energy bond between an amino acid and adenosine monophosphate (AMP) a pyrophosphate molecule is expelled in this reaction. The activated amino acid is then transferred to the tRNA, and AMP is released. The term "charging" is appropriate, since the high-energy bond that attaches an amino acid to its tRNA is later used to drive the formation of the peptide bond. Each tRNA is named for its amino acid.
The Mechanism of Protein Synthesis
As with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. The process of translation is similar in prokaryotes and eukaryotes. Here we’ll explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between prokaryotic and eukaryotic translation.
Initiation of Translation
Protein synthesis begins with the formation of an 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 tRNA Metf .
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. Guanosine triphosphate (GTP), which is a purine nucleotide triphosphate, acts as an energy source during translation—both at the start of elongation and during the ribosome’s translocation. Binding of the mRNA to the 30S ribosome also requires IF-III.
The initiator tRNA then interacts with the start codon AUG (or rarely, GUG). This tRNA carries the amino acid methionine, which is formylated after its attachment to the tRNA. The formylation creates a "faux" peptide bond between the formyl carboxyl group and the amino group of the methionine. Binding of the fMet-tRNA Metf is mediated by the initiation factor IF-2. The fMet begins every polypeptide chain synthesized by E. coli, but it is usually removed 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 Met . After the formation of the initiation complex, the 30S ribosomal subunit is joined by the 50S subunit to form the translation complex. In eukaryotes, a similar initiation complex forms, comprising mRNA, the 40S small ribosomal subunit, eukaryotic IFs, and nucleoside triphosphates (GTP and ATP). The methionine on the charged initiator tRNA, called Met-tRNAi, is not formylated. However, Met-tRNAi is distinct from other Met-tRNAs in that it can bind IFs.
Instead of depositing at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 7-methylguanosine cap at the 5' end of the mRNA. A cap-binding protein (CBP) and several other IFs assist the movement of the ribosome to the 5' cap. Once at the cap, the initiation complex tracks along the mRNA in the 5' to 3' direction, searching for the AUG start codon. Many eukaryotic mRNAs are translated from the first AUG, but this is not always the case. According to Kozak’s rules , the nucleotides around the AUG indicate whether it is the correct start codon. Kozak’s rules state that the following consensus sequence must appear around the AUG of vertebrate genes: 5'-gccRccAUGG-3'. The R (for purine) indicates a site that can be either A or G, but cannot be C or U. Essentially, the closer the sequence is to this consensus, the higher the efficiency of translation.
Once the appropriate AUG is identified, the other proteins and CBP dissociate, and the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit. This step completes the initiation of translation in eukaryotes.
Translation, Elongation, and Termination
In prokaryotes and eukaryotes, the basics of elongation are the same, so we will review elongation from the perspective of E. coli. When the translation complex is formed, the tRNA binding region of the ribosome consists of three compartments. The A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. The initiating methionyl-tRNA, however, occupies the P site at the beginning of the elongation phase of translation in both prokaryotes and eukaryotes.
During translation elongation, the mRNA template provides tRNA binding specificity. As the ribosome moves along the mRNA, each mRNA codon comes into register, and specific binding with the corresponding charged tRNA anticodon is ensured. If mRNA were not present in the elongation complex, the ribosome would bind tRNAs nonspecifically and randomly.
Elongation proceeds with charged tRNAs sequentially entering and leaving the ribosome as each new amino acid is added to the polypeptide chain. Movement of a tRNA from A to P to E site is induced by conformational changes that advance the ribosome by three bases in the 3' direction. The energy for each step along the ribosome is donated by elongation factors that hydrolyze GTP. GTP energy is required both for the binding of a new aminoacyl-tRNA to the A site and for its translocation to the P site after formation of the peptide bond. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase , an RNA-based enzyme that is integrated into the 50S ribosomal subunit. The energy for each peptide bond formation is derived from the high-energy bond linking each amino acid to its tRNA. After peptide bond formation, the A-site tRNA that now holds the growing peptide chain moves to the P site, and the P-site tRNA that is now empty moves to the E site and is expelled from the ribosome (Figure 15.16). Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino-acid protein can be translated in just 10 seconds.
Many antibiotics inhibit bacterial protein synthesis. For example, tetracycline blocks the A site on the bacterial ribosome, and chloramphenicol blocks peptidyl transfer. What specific effect would you expect each of these antibiotics to have on protein synthesis?
Tetracycline would directly affect:
Chloramphenicol would directly affect:
Termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered. Upon aligning with the A site, these nonsense codons are recognized by protein release factors that resemble tRNAs. The releasing factors in both prokaryotes and eukaryotes instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. This reaction forces the P-site amino acid to detach from its tRNA, and the newly made protein 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
During and after translation, individual amino acids may be chemically modified, signal sequences appended, and the new protein “folded” into a distinct three-dimensional structure as a result of intramolecular interactions. A signal sequence is a short sequence at the amino end of a protein that directs it to a specific cellular compartment. These sequences can be thought of as the protein’s “train ticket” to its ultimate destination, and are recognized by signal-recognition proteins that act as conductors. For instance, a specific signal sequence 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.
Many proteins fold spontaneously, but some proteins require helper molecules, called chaperones, to prevent them from aggregating during the complicated process of folding. 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.