Translation, Part 2

     Who we are in both our mental and physical attributes begins with the expression of our genetic heritage. Our genes must express themselves to provide us with a physical structure (our anatomy) and with functionality and regulation for our parts (physiology). Gene expression starts with the code that is the sequence of bases composing the DNA molecules locked inside the nuclei of our cells. But gene expression requres the translation of that sequence of bases in DNA into a sequence of amino acids in proteins. Those proteins, tens of thousands of different ones, are organized into our living cells and express our genes through their structure and actions. This is the premiere process occurring in cells; cells devote more energy to protein synthesis than to any other aspect of metabolism. A typical bacterial cell (e.g., E. coli) contains about 200,000 ribosomes. Eucaryotic cells (like yours) contain even more of them. This is no surprise. Our proteins, interacting with other macromolecules, are the genetic basis of what and who we are.

Transcription

     The first step of translation occurs in the nucleus and is the transcription of DNA's code onto molecules of messenger RNA (m-RNA). We considered this process of transcription in the previous exercise of this WebLab. Then the m-RNA molecules must leave the nucleus, enter the cytoplasm of the cell, and associate with ribosomes. Ribosomes are the protein synthesizing factories of cells. But where did the ribosomes come from? They are constructed from ribosomal RNA (r-RNA) molecules (also constructed from DNA templates in the cell's nucleus) and proteins.

Amino Acids are carried by t-RNA

     After we imagine m-RNA molecules asociated with ribosomes ready to produce proteins, we must ask: where do the amino acids come from and how do they get to the ribosomes? Amino acids come from the proteins we eat. We digest those proteins into amino acids and absorb them into our blood from the small intestine. Bacterial cells would get amino acids by absorbing them from the liquid or solid meduim on which they are living. Once transported through the cells outer membrane (called the cell membrane or plasma membrane) the amino acids enter the cell's cytoplasm. Then the amino acids attach to transfer RNA molecules (t-RNA) that carry individual amino acids to the ribosomes. Transfer RNA molecules are also constructed from DNA templates in the nucleus.

Codon-Anticodon Match-up

     Another important question is: how are the amino acids placed in the precise order required by the genetic code? In other words, how is the primary structure of a protein (polypeptide) insured to come out correctly? These details are handled by the t-RNA molecules as they link up with the m-RNA molecules on the ribosomes. Each three bases of m-RNA is called a codon. Each codon specifies, or stands for, a distinct amino acid. The t-RNA molecules possess anticodons. These are base sequences complementary to the genetic code's codons. Codon-anticodon match-up insures that each t-RNA aligns itself at the right spot along the m-RNA molecule. This insures that the correct amino acid gets into the right spot in the growing polypeptide chain.

Some Details of the Cellular Components Used for Protein Synthesis
The Ribosomes
Ribosomal Size

     Ribosomes found in all cells are composed of approximately 50% ribosomal RNA and 50% protein. They all have the same basic three dimensional structure. There are just differences in overall size. The largest ribosomes come from eucaryotic cells (these are the cells from all the kingdoms of living organisms except the two bacterial kingdoms - the Archaebacteria and the Eubacteria). Ribosomes from procaryotic cells (the two bacterial kingdoms) are slightly smaller than eucaryotic ribosomes. The smallest ribosomes are found inside mitochondria and chloroplasts (that these organelles possess their own ribosomes might come as a surprise to you if you don't already know about the theory of endosymbiosis; you could look up the word!).

Polyribosomes and m-RNA

     Each ribosome consists of two pieces, each one looking roughly like an ellipse, that come together just prior to protein synthesis. The two pieces separate just after protein synthesis is complete. Ribosomes work together in groups called polyribosomes. A messenger RNA molecule threads itself along a groove at the junction between the two ribosomal subunits. Then, as it is being "read" or translated by that ribosome, it emerges out from that ribosome and threads itself into the next ribosome, and so on. In this way, several ribosomes are translating the genetic code on the messenger simultaneously, and each ribosome produces a copy of the specified protein. Very efficient! To visualize this process, go to The Control of Gene Expression and scroll down to the second illustration which consists of three images - OBSERVE THE MIDDLE IMAGE and look for the structures labeled m-RNA, ribosome, 1st polypeptide, and ribosomal subunits. Ignore the other labels.
     The first ribosome threaded into by the messenger finishes translation first and the completed protein drops off and the ribosome's two components separate. Then the second ribosome that the messenger associated with finishes translation, and its protein drops off and that ribosome's components separate. And so on.

The Genetic Code of Messenger RNA
64 Possible Codons

     Translation is the process of converting a sequence of bases in DNA into a sequence of amino acids in a protein. The sequence of bases transcribed onto m-RNA, and delived to the ribosome as the m-RNA molecule, is called the genetic code. There are 4 bases used in m-RNA - adenine, guanine, uracil, and cytosine - and the genetic code is a triplet code, meaning that each sequence of 3 bases codes for one amino acid. Each group of 3 bases coding for one amino acid is called a codon. So the first base of the codon can be any one of the 4 possibilities. Similarly the second base of the codon can be any one of the 4 bases. And finally, the third position of the codon can be any one of the 4 bases. This is expressed mathematically as 4 to the third power, or 4³. This is computed as 4 x 4 x 4 which equals 64. What this means is that there are 64 possible codons.

Start and Stop Codons

     Experiments have proven that 61 of the codons are specific for amino acids. The remaining 3 codons signal polypeptide chain termination (in other words, stop translation and let the completed protein fall off the ribosome). Interestingly, the codon for the amino acid, methionine, is the signal to "start" translation. This means that every protein starts out having methionine as its first amino acid. However, after translation is completed, the methionine is often removed in what is called post-translational modification.

The Code is Degenerate

     Another aspect of the genetic code is that most amino acids are coded for by several different codons. Actually, only two amino acids have just one codon that represents them. This is expected since the genetic code specifies only 20 amino acids, and yet there are 61 functional codons. So some amino acids must be coded for by more than one codon! Because of this fact, the code is said to be degenerate. You can verify this by re-looking at the What is a Gene Translation Site and scrolling down to the table presenting the genetic code. Find out which are the two amino acids that have only one codon representing them.

Ribosomal RNA
     Recall that the ribosomes are composed of about 50% protein and 50% RNA. The type of RNA in the ribosome is called specifically ribosomal RNA (r-RNA). In eucaryotic cells r-RNA is synthesized in specific regions of the nucleus called the nucleolus. This area appears as a dark spot or several spots within the nucleus. The nucleolus (or nucleoli) is a region of the nucleus where there are many copies of ribosomal RNA-coding genes. After the r-RNA is synthesized, it is modified and constructed into ribosomal subunits (recall that each ribosome is composed of two subunits). The subunits can then migrate out from the nucleus into the cytoplasm where they can associate with each other to become complete ribosomes - the cellular factories for protein synthesis.

Transfer RNA
     Transfer RNA molecules carry amino acids to the ribosomes where they can be linked together in the proper sequence to form polypeptides. Transfer RNA molecules possess truly precise and extensive specificity. Take a three dimensional look at a transfer RNA molecule by going to The 3D t-RNA Site.

t-RNA Must Bond to the Correct Amino Acid

     Transfer RNA molecules must be recognized by the correct enzyme (called an aminoacyl t-RNA synthetase) so they can be loaded with the correct amino acid.
     Just imagine the cytoplasm of a cell where tens of thousands of amino acid molecules are located. A given t-RNA must locate and bind the specific one of the 20 different amino acids that it is constructed to pick up. How can this happen? On the surface of an enzyme specific for that purpose, of course! So there is a different aminoacyl t-RNA synthetase enzyme for each different t-RNA molecule and the amino acid to which it binds. So the specific shape of the t-RNA must conform to a specific shape on the enzyme. Then the enzyme works in two steps. First the amino acid is activated using a molecule of ATP (the cell's most commonly used high energy molecule), and then the activated amino acid is attached to the t-RNA molecule. The resulting molecule is represented as t-RNA~aa (It is called an aminoacyl-t-RNA. The squiggly line between the t-RNA and the aa (amino acid) means that the bond is a high energy bond.

t-RNA Must Bond to the Correct Codon on m-RNA

     Transfer RNA molecules possess the correct anticodon so that, upon reaching the ribosome, they can align with the proper codon on the messengeg RNA. This assures that the amino acid carried by that t-RNA will be added to the growing polypeptide in the correct sequence as coded for in the sequence of m-RNA bases. Since there are 61 codons that actually code for amino acids, there are 61 anti-codons found on the different types of t-RNA molecules.

t-RNA Must Bond to the Correct Spot on the Ribosome

     Transfer RNA molecules bind to the correct spot on the ribosomal surface. Compared with the size of an individual t-RNA molecule, a ribosome is much larger. The t-RNA must "dock" itself into the correct spot. This requires a complementary shape so that there is correct placement and alignment with respect to both the m-RNA molecule and the previously attached t-RNA.

t-RNA Molecules as "Adaptors"

     Overall, what we see are transfer RNA molecules acting as "adaptor" molecules. The t-RNA's are the intermediaries between the genetic code, on the m-RNA threaded along the ribosomes, and the amino acids that each t-RNA is carrying. The anticodon area of the t-RNA and the overall, specific shape of the t-RNA determines its fit onto the ribosome at the exact moment that the amino acid it is carrying is called for. When you consider that multiple copies of proteins containing 300 hundred to a thousand amino acids can be synthesized typically in under a minute, the efficiency of these processes is truly astounding.

Translation
Review of the Components for Protein Synthesis

     The previous discussion has presented details of the components of cells used for protein synthesis: m-RNA carrying the nuclear DNA-derived genetic code for how to build a protein, ribosomes as the cytoplasmic sites for protein synthesis, and t-RNA molecules carrying amino acids to be incorporated into synthesizing polypeptide chains, and possessing anticodons to match up with the m-RNA codons.

Initiation

     The first step in translation is initiation. The m-RNA from the nucleus attaches to the smaller of the two ribosomal subunits. Then the anticodon of the first t-RNA~aa attaches to the start, or initiation, codon (always AUG) of the messenger RNA. Initiation is completed as the larger ribosomal subunit is attached to the smaller subunit. All these initial "attachments" require special protein initiation factors and an energy source. The energy source is, in this case, a compound related to ATP - it is called GTP (guanosine triphosphate). So at the end of initiation, we have 1) a complete ribosome (both subunits have come together), 2) the m-RNA is in place threaded along the ribosomal surface, and 3) the first t-RNA is already attached. Remember that the first t-RNA is always the same: the signal to "start" a protein is the codon AUG, which binds to the t-RNA anticodon, UAC, which is always on a t-RNA carrying methionine.

Elongation

     Before beginning to read this section take a look at this Translation Diagram from "To Know Ourselves" by the U.S. Dept.of Energy. You will then have a visual image of what is being described.
      The second translational step is called elongation. This is a step that will repeat itself as many times as is necessary to complete the synthesis of the polypeptide. With each round of the elongation process, one additional amino acid is added to the growing polypeptide.
     t-RNA binds to the ribosome  The first step in elongation is a t-RNA~aa binding to the ribosome right next to the preceding t-RNA~aa. Which t-RNA binds second depends on the codon right after the initiation codon. So the second t-RNA~aa now binds to the ribosome and that t-RNA is parked right next to the first (initiator) t-RNA.
     peptide bond formation   At this point, the second step of elongation is the formation of a peptide bond between the first and second amino acids that are positioned right next to each other. This peptide bond formation requires an enzyme, peptidyl transferase, and an energy source which is GTP.
     ribosome shifts its position   The last step in elongation is the physical shifting of the ribosome along the m-RNA molecule to align the next codon of m-RNA for binding to the anti-codon of the next (in this case the third) t-RNA. This physical shifting of the ribosome along the m-RNA requires the energy of GTP. In addition, elongation requires special proteins called elongation factors.
     So at the end of the first round of elongation, can you picture the ribosome? It has the m-RNA threaded in it, and there are two t-RNA's attached to it: the initial t-RNA that always codes for methionine, and the second t-RNA which codes for the second amino acid, which is whatever amino acid was called for in the second codon of the m-RNA. Which t-RNA is holding the dipeptide, which is as much of a polypeptide that has been synthesized so far? The second t-RNA! And at the end of the third round of elongation, the third t-RNA that has attached to the ribosome will be holding the tripeptide, and so on.

The Forming Polypeptide Assumes its Secondary and Tertiary Structure

     As each amino acid is added to the growing chain, the t-RNA molecules that have given up their amino acids drop off the ribosome and can be used again to pick up other amino acids. As the polypeptide elongates (from a dipeptide, to tripeptide and up to however long the polypeptide is coded for by m-RNA) it spirals and pleats (secondary structure), and folds (tertiary structure) into its natural three dimensional conformation. It does this right on the surface of the ribosome as the polypeptide is still forming. In the bacterium E. coli, amino acids are added at a rate of about one each 0.05 seconds, or at a rate of about 500 amino acids (the size of a typical protein) per 25 seconds.

Termination

     The third translational step is called termination. It occurs when any of the three termination codons (UAA, UAG, or UGA) shows up along the m-RNA as it is read on the ribosome. The termination codons are recognized by release factors which add a water molecule to the previous amino acid, instead of another amino acid. Now the m-RNA detaches from the ribosome, the polypeptide drops off the ribosome, and the ribosome separates into its two subunits. The two ribosomal subunits are now ready to begin a new round of protein synthesis repeating the above steps.

RETURN TO WEBLAB#5: EXERCISE#2