The Atelier and Gene Pool
The Gene Pool
Natural Selection in Microorganisms:
If you walk right into the water of the Gene Pool, you will find the Natural Selection activity. The activity starts with a box that generates an instructional notecard and 6 microorganisms: 3 red and 3 green. The student acts as the selective agent by clicking on each of the microorganisms. They will either reproduce or die, depending on their individual resistance. One group is more resistant than the other. As the microorganisms reproduce, the student clicks on all of the individuals produced in each generation. After the fifth generation, the number of each type of microorganism remaining is counted.
A series of giant ribosomes are lined up near the Gene Pool, illustrating the stages of translation in protein synthesis. In addition to the three translation models, a fourth ribosome serves as a platform where students, acting as tRNAs, can hop on and off the ribosome’s tRNA binding sites as they build the insulin precursor.
Three stages of translation are numbered, to indicate the appropriate order of study. There are multiple active elements in these models, including several informational signs, the ribosomal subunits, the mRNA, tRNAs at each stage, and the tRNA binding sites on the ribosomes
Protein Synthesis Sign:
Clicking on this sign delivers a notecard that briefly describes the role of proteins in cell function and notes that the 3-dimensional conformation of a protein depends on its constituent amino acids being put into the correct sequence. This sequence is encoded in DNA. However, the information of DNA is carried from the chromosome to ribosomes by an intermediary molecule: messenger RNA (mRNA).
Differences between RNA and DNA are summarized: RNA is single stranded rather than duplexed, and can form internal base pairs to fold into complex shapes. RNA contains the sugar ribose instead of the DNA sugar deoxyribose, and its fourth base is uracil rather than the thymine found in DNA. Uracil has the same base pairing properties as thymine, base pairing with adenine.
The notecard also directs you to a second sign that explains the function of another RNA necessary for translating genetic messages: tRNA.
Although the ribosome is the largest and most conspicuous player in the process of protein synthesis, the critical element in the process of translating the genetic code is tRNA or transfer RNA.
Transfer RNAs are a family of small RNAs containing about 80 nucleotides. Like most RNA molecules, they are single stranded and can fold into complex forms by localized regions of base pairing (A=U, G=C). In addition to the "typical" RNA bases, tRNA also contains a number of unusual bases like inosine, ribothymine, and pseudouracil. The regions of RNA base-pairing divide tRNAs into four regions: the amino acid binding site, the T-Loop, the D Loop and the Anticodon Loop.
(Redrawn from http://www.med.unibs.it/~marchesi/protsyn.html)
- The amino acid binding stem contains both the 5' and 3' ends of the molecule, base paired, but with the ends offset by 3 nucleotides, so that the 3' end of all transfer RNAs consists of 3 unpaired nucleotides: C-C-A. The amino acid binds to the terminal A nucleotide. Binding an amino acid to tRNA requires energy supplied by ATP.
- The Anticodon Loop is located opposite the amino acid binding site, and includes a 3-base anticodon, which is complementary to specific codons on the mRNA. Codon-anticodon base pairing allows specific amino acids to be lined up opposite specific mRNA codons
- The T and D loops are both named for unusual bases within the loops, and in the 3-dimensional folding of the molecule, are close together.
Translation Stage 1: Initiation
This stage involves the assembly of the initiation complex consisting of mRNA, the small ribosomal subunit, and the initiating tRNA. Each of these objects is interactive and will deliver an informational notecard explaining its role in translation.
The mRNA represented here displays the codons for the first 8 amino acids of the polypeptide precursor for the peptide hormone insulin. The full mRNA has 333 nucleotides, or 111 codons, including the start codon for the amino acid methionine and the stop codon UAG. The encoded polypeptide is 110 amino acid insulin precursor, from which two shorter peptide chains are snipped out and then combined to form the active hormone.
Small Ribosomal Subunit:
Ribosomes are complex macromolecules consisting of two subunits. Each of the two subunits contains both RNA and protein. The small subunit contains a single ribosomal RNA (rRNA) and a number of proteins. The rRNA is folded by local regions of base pairing into a complex form that is nested within the ribosomal proteins.
Protein synthesis is initiated when the initiating tRNA, usually a tRNA for the amino acid methionine, is attached to its amino acid. The methionine + tRNA then binds to the small ribosomal subunit. After the methionine-tRNA is bound, mRNA can bind to the ribosome, and is aligned so that the methionine codon AUG is matched to the tRNA anticodon. Binding to the ribosome is an energy requiring process, and also uses several protein initiation factors.
Stage 2: Peptide Bond Formation
After formation of the initiation complex, the large ribosomal subunit is added to make the translation complex. This complex contains the binding sites for 3 tRNAs and catalyzes the formation of the peptide bond that connects amino acids.
To form the Translation Complex, the initiation complex binds the large ribosomal subunit. The large ribosomal subunit has two rRNAs (one large and one small) plus about 30 (in prokaryotes) to 50 (in eukaryotes) proteins. Eukaryotic ribosomes have an additional small rRNA in the large subunit.
The translation complex includes three binding sites for tRNA: the A site, the P site and the E site.
Peptide Bond Formation:
Amino acids have two defining functional groups: the amino (NH2) group and the carboxyl (COOH) group. These are the same in all amino acids. The “R” group, which represents one of 20 different functional groups, makes one amino acid different from another. When two or more amino acids are linked together, a peptide bond is formed by connecting the carbon of the carboxyl group directly to the nitrogen of the amino group.
Because peptide bond formation is a chemical reaction, ribosomes have catalytic activity. But which of the ribosome’s constituents is the catalytic site? Ribosomes contain many proteins, but none of them is close enough to the site of peptide bond formation to be the catalyzing molecule. It is possible that the catalytic activity resides in ribosomal RNA. Other catalytic RNAs, called ribozymes, are known.
tRNA binding sites:
- A Site: The A site is where the second and all following tRNAs, carrying their amino acids, will bind. Click on the two tRNAs to find out more about each.
- P Site: The P site of the ribosome binds the initiating tRNA. Only this initiating tRNA goes straight to the P site. Other tRNAs bind first to the A site.
- The E site of the ribosome binds to tRNA from which an amino acid has been removed.
The codon for Methionine, AUG, is located over the P site. Amino acids are attached to tRNA by their carboxyl ends. Aminoacyl-tRNAs are high energy molecules, and a lot of energy is released when an amino acid is removed from its tRNA. Much of the energy required for making the peptide bond between amino acids comes from the removal of an amino acid from its tRNA.
After the initiation complex is formed, all new tRNAs bind to the A site. The tRNA in the A site on this ribosome is for the amino acid alanine. The anticodon of the tRNA recognizes the mRNA codon. Binding tRNA to the A site of a ribosome requires energy (provided by the high energy molecule GTP) and is assisted by a protein elongation factor. The Methionine carried by the tRNA at the P site will be released from its tRNA and attached to the amino acid on the second tRNA by a peptide bond. The Alanine-tRNA will then be carrying two amino acids: Met-Ala-tRNA.
After the peptide bond has formed both tRNAs will move or translocate. The empty tRNA at the P site will move to the E site. The Met-Ala-tRNA will move to the P site. Then the next tRNA will bind to the A site.
Stage 3: Elongation
All three tRNA-binding sites are now occupied by a tRNA. This state of the ribosome will continue as the mRNA advances, one codon at a time, through it. Click on each of the binding sites for additional information.
tRNA binding sites:
- A Site: The third amino acid, on its tRNA, has now bound to the A site.
- The P site of the ribosome now serves the second of its two functions. After formation of a peptide bond between two adjacent amino acids, the tRNA carrying the new amino acid and the growing peptide chain moves into this site.
- The E site of the ribosome binds to tRNA from which an amino acid has been removed.
The tRNA now bound to the A site carries leucine, the next amino acid to be added to the protein chain. The anticodon of the tRNA recognizes the leucine codon CUG, which corresponds to the amino acid that it carries. Some tRNAs can recognize more than one mRNA codon for the same amino acid, but all tRNAs are specific for a single amino acid.
After the initiating tRNA, all other tRNAs migrate to the P site from the A site. These tRNAs will carry their original amino acid plus whatever amino acids have become attached to it by peptide bond formation. On this ribosome is an alanine tRNA carrying the amino acid alanine. However, the initiating amino acid methionine is now attached to the alanine. This Met-Ala dipeptide (two amino acids) will be attached to the new amino acid on the A site, in this case leucine. What will the resultant tripeptide look like?
Once tRNAs have given up their amino acid, they move into the E site of the ribosome. This is an Exit platform from which the tRNA will be released from the ribosome on the next reaction cycle.
Stop Sign: Polypeptide Termination
What happens with one of the three stop codons is pulled into position over the A site? There is no transfer RNA that corresponds to any of the stop codons: UAG, UAA, UGA. At the A site, a protein termination factor binds instead a transfer RNA. Instead of transferring its peptide chain to a new amino acid, the tRNA at the P site passes the carboxyl end of its bound amino acid to a water molecule, completing the COOH carboxyl group.
With the last tRNA freed, the linchpin that holds the translation complex together is removed. The peptide chain is freed, the tRNAs are released, and the two ribosomal subunits separate.
The Translation Game:
On this giant ribosome, students can play the role of tRNA, carrying amino acids to the ribosome and connecting them to form a multiunit protein. The mRNA codons for the insulin precursor are loaded into the ribosome and will advance when clicked. tRNAs will match themselves to the appropriate codon and then pick up the growing chain of amino acids.
Ideally, the game should be played with enough individuals to represent tRNAs for each of the amino acids. However, in smaller groups, a single student can play multiple tRNA roles. Students should be given copies of the amino acid or amino acids for which they will be responsible.
The game will work something like this, although once students start playing, wild modifications may ensue!
- The person acting as the initiating Met-tRNA will take a copy of the amino acid Methionine out of the amino acid bowl to the right of the coffee shop. The copy should appear in your inventory.
- The next person will take the amino acid corresponding to the codon over the A site. As new codons appear over the A site, the next tRNA player takes a copy of that amino acid from the bowl.
- The first two players take their places by sitting on the appropriate binding sites.
- When both tRNAs are in place, the initiating tRNA pulls his copy of Methionine out of inventory and drops it into the Peptide Chain trough between the A and P sites. The amino acids are physical and will roll to the end of the trough.
- The tRNA on the P site then moves to the E site and clicks on the codon chain to advance to the next codon. AUG should now be over the E site and UUG over the P site. A new codon will appear over the A site. The tRNA #3 then sits on the A site, and tRNA#2 pulls his or her amino acid out of inventory and drops it into the Peptide trough.
After tRNA#2 drops his amino acid, he or she moves to the E site, replacing the initiating tRNA. As each player moves into the E site, the codon chain should be clicked to advance to the next codon.
Continue the cycle of picking up the amino acid indicated by the A site codon, advancing to the P site, and then dropping your amino acid into the trough.
When one of the three stop codons appears above the A site, the end of the message is signalled. The tRNA on the P site drops his or her amino acid into the trough to complete the peptide.
If all has gone well, the amino acids should be lined up in order in the peptide trough. Walk back and read the message from left to right!
Amino acid names are abbreviated using either three letter designations, like Met, Phe, and Ala or single letter designations like M, F and A. The single letters spell out the message.
- When you get good at this, consider making a movie like the one linked to the ribosome!
The Atelier is the “sandbox” or working area for student projects or other people that want to contribute an interactive object to Genome Island. The sandbox is a multilevel structure, and includes four offground platforms in addition to the ground platform down the hill from the Abbey. These provide ample working space for building new objects. Students and colleagues can be invited to join the Genetics Atelier, which gives them building privileges in this area of the island. Completed projects are integrated into the activities offered at Genome.Genome has benefitted from the collaborative efforts of many individuals, including the following:
- Susan Tzuki: Susan suggested and assembled the links for the Evolutionary Genetics library in the Tower near the Fly Lab.
- Elizabeth Gloucester: Elizabeth, who teaches and directs research in microbiology at a medical school in New England, wrote the notecards for the Garden of Prokaryotic Genomes.
- Apaul Balut: Apaul, who teaches and directs research in microbiology at the medical school of Wayne State University, added valuable information to the notecard on human mitochondrial genomes in the Tower.
- Panaceah Omegamu: Panaceah created and gifted me with the beautiful DNA model now displayed in the DNA area of the tower. It was much prettier than the one I had build myself!
Since she kindly gave me modification permission I was able to add the ATCG symbols for the paired bases of DNA showing the base pairing and also the size difference between purines and pyrimidines.
- Timothy Trenchmouth: Timothy created and gave me the very nice models of the four DNA bases also displayed in the DNA area of the tower. When all four bases are rezzed, they or orientated in their base-pairing positions.
- Ambrosia Lytton: Ambrosia and her team are developing The Bunny Hutch, which will illustrate many principles of gene interaction in the inheritance of coat color and pattern in rabbits.
- Clowe Greenwood: Clowe and Apaul will be collaborating on an activity for the Gene Pool: a game based on the development of antibiotic resistance in bacterial populations.
- Poulet Collas: Poulet and his friend and colleague Catus are working on models to link Second Life objects to internet bioinformatics software, especially for the study of prokaryote genomes.
- Catus Milos: See entry for Poulet Collas above.
- Yoghas Etchegaray: Yoghas is an MD interested in the genetics of human disorders and will be adding a Human Diseases section to the Human Genetics area in the tower.
- Graham Mills: Graham is working on an interactive model to explain genetic regulation.
- Asorel Todriya: Asorel has created several models of 3-D protein structure. Only the backbone is displayed to reduce prim load. Asorel’s Green Fluorescent Protein hangs outside of the bacterial transformation lab in the Tower, casting an eerie greenish light from its chromophore. Asorel has other molecular models on display over at the Science Center.