T5l1m3 translation transcriptCloverleaf model has three bases; these are unique to each tRNA type.
Amino acids are always attached to the 3site opposite from the anticodon region.
The cell cytoplasm is full of transfer RNA molecules and free amino acids waiting to be assembled into polypeptides.
If the cytoplasm is full of free amino acids and transfer RNA molecules how does the correct amino acid match up with the appropriate tRNA?
This is accomplished by the enzyme aminoacyl-tRNA synthetase; a specific version of this enzyme is present in the cytoplasm for each amino acid.
As indicated in the diagram, this enzyme has an amino acid site, an ATP site and a tRNA site.
The enzyme catalyzes a reaction between ATP and the correct amino acid thus activating the amino acid.
The appropriate tRNA can now be accepted by the enzyme and the tRNA and amino acid are covalently bound together.
The enzyme releases the aminoacyl tRNA which is now ready to transport a specific amino acid to the site of polypeptide assembly, the ribosome.Slide 8
Before we can discuss the process of translation, we first need to look more closely at the ribosome.
A ribosome is made up of a large subunit and a small subunit which only come together during translation.
When the ribosome is not interpreting mRNA the subunits remain separate in the cytoplasm.
Each subunit is composed of ribosomal RNA and proteins.
Ribosome subunits are produced in the nucleolus of eukaryotic cells and then exported to cytoplasm.
A ribosome is not associated with just one type of protein; it can transcribe many different mRNA molecules making many different polypeptides.The genetic information on the mRNA is translated at specific sites on the ribosome.There are four docking sites associated with the large subunit: T, A, P, E.
The transfer (T) site is where the activated amino acid arrives at the ribosome accompanied by a transfer factor.
The amino acid (A) site is where the tRNA anticodon pairs with the mRNA codon holding the next amino acid to be added to the chain.At the polypeptide (P) site the tRNA holds the amino acid that is being incorporated into the polypeptide chain.
Once the amino acid is incorporated into the growing polypeptide chain, the tRNA is discharged through the exit (E) site.
Note that the tRNA and mRNA only interact at the A and P sites on the ribosome.Slide 9
The synthesis of a polypeptide can be divided into three steps.
The first step, initiation, occurs when the ribosome subunits and mRNA come together; elongation is the second step and occurs when amino acids are added to the growing polypeptide chain; termination is the final step when a stop codon ends translation of the polypeptide and it is released from the ribosome.
Now lets look at the events that occur during each stage.Slide 10 The small subunit of the ribosome recognizes a specific sequence on the mRNA that is adjacent to the start codon and binds to it.
The tRNA with the first amino acid of the polypeptide matches up with the start codon AUG.
The first amino acid is always methionine.
The large subunit of the ribosome joins the initiation complex.
Once this has occurred the active tRNA is docked in the P site of the large subunit.
The A site is now ready to accept the next tRNA and begin building the polypeptide.Slide 11 The polypeptide chain becomes longer, one amino acid at a time.
The next active tRNA docks in the A site.
In this example it carries the amino acid proline.
The large ribosome subunit catalyzes the formation of a peptide bond between the two amino acids.
The free tRNA exits the ribosome complex and the ribosome shifts over one codon.
The tRNA with proline and methionine now occupies the P site leaving the A site vacant.
Another aminoacyl tRNA with an anticodon matching the mRNA docks in the A site.
The amino acid is added to the growing polypeptide through formation of another peptide bond.The ribosome again shifts one codon and the tRNA exits from the E site.
This continues until the polypeptide reaches its designated length.
Notice that during translation the 5end of the mRNA moves through the ribosome first.
How long does it take to make a polypeptide chain, you ask?
Ten or more amino acids may be added per second!
Considering the mechanics and biochemical reactions that are involved, the process of translation is very rapid.Slide 12 The growing polypeptide is complete once a stop codon in the mRNA either UAA, UAG or UGA - enters the A site
A protein release factor binds to the stop codon in the A site and a water molecule is added to the polypeptide in place of an amino acid causing it to be released from the ribosome.
All the components associated with the ribosome disassemble and return to the cytoplasm as individual components.Slide 13 Once the newly formed polypeptide is released from a ribosome it folds into a three dimensional shape depending on the amino acid sequence of the protein.Towards the end of translation signal peptides are incorporated into the molecule.
These signal peptides provide a specific instruction that determines if the polypeptide is to complete translation in the endoplasmic reticulum, or translation is to be completed in the cytoplasm.
If completed in the cytoplasm, signal peptides direct the protein to specific organelles in the cell.Slide 14 In order to accommodate the high turnover of proteins in a cell, many polypeptides can be synthesized from the same strand of mRNA at the same time.
This chain of actively translating ribosomes is called a polysome or polyribosome..
Betaglobin211- 1 --globin (Hemoglobin) Gene SequenceBeta-globin (globin) is one of the protein subunits that make up the protein, hemoglobin.Hemoglobin is a quaternary protein comprised of two alpha subunits linked with two betasubunits.Each subunit binds a heme group, which in turn binds one iron atom (Fe).Oxygen canbind reversibly to this Fe atom.As a red blood cell passes through the lungs, oxygen diffusesinto the cell and binds to the Fe of the heme group on all four subunits of the hemoglobintetramer.A common form of sickle-cell anemia is caused by a single point mutation in the nucleotidesequence of -globin.The mutation is located in the seventh codon (The first codon codes forMet, the leader amino acid in polypeptides.) The seventh triplet should read GAG which coldsfor glutamic acid, but the middle nucleotide has changed to a thymine, which changes the tripletto GTG, which codes for valine.globin's three-dimensional structure places this seventhamino acid on the outside of the protein.
Replacement of the normally charged glutamic acidwith the hydrophobic valine alters the solubility of hemoglobin, so that at a lower oxygenconcentration, the altered protein changes the red blood cell to a sickle shape that is unable toIn this activity you will be observing a portion of the actual nucleotide sequence for the polypeptide.This sequence (found at the end of this exercise) was obtained from GenBank, aninternet source, under the key word: HUMHBB.
There are actually 73,308 nucleotides forhemoglobin, which is located at the tip of chromosome 11.
This includes the adult globin geneas well as several other related globin genes (fetal, -globin, etc.).
The entire hemoglobinnucleotide sequence takes 40 pages to list.Taped
together in the correct order, it wouldrepresent just 0.002% of the entire human genome.
The sequence that contains the information is located between nucleotides 62,137
63,660.You will be tracing the information flow from DNA
amino acid sequence of theglobin polypeptide in this exercise..
Ucm088974510(k) Summary TABLE OF CONTENTS Page 1 of 7 I T------ 2.Intended Use / Indications for Use 3.Device Description HIV-l HIV-I 4.Potential Risks associated with use of the Device V,tro HIV-1 Genotyping Kit and OpenGene DNA sequencing system.There have been reportable events currently documented for this device.Potential user hazards were evaluated and the device hardware (Long-Read Tower Sequencer and Gel Toaster Polymerization unit) met the following safety standards: Certificate of Compliance, Certificate Number LR 109279-6 l CAN/CSA-C22.2 No.1010.1-92 Safety Requirements for Electrical Equipment for Measurement, Control and Laboratory Use, Part I: General Requirements (Includes Amendment 1) Page 4 of 7 l CAN/CSA-C2.2 No.lOlO.lB-97 Amendment 2 to CAN/CSA-C22.2 No.1010.1-92 Safety Requirements for Electrical Equipment for Measurement, Control and Laboratory Use, Part I: General Requirements l l IEC Publication 61010-l Edition 1:1990, including Amendment No.1 (1992) and Amendment No.
2 (1995), conformity testing and certification of electrical equipment Verification Certificate for Electromagnetic Compatibility Requirements File No.VIS-014- IEC3-2 .
IEC lOOO-3-2/EN61000-3-2 H armonic Current emissions (Input current
16 Amps per Phase) l 6.Clinical Studies Table 1 gives a summary overview of the clinical studies, and the results of the studies for base calling accuracy, codon agreements, sensitivity and specificity measures of the device.Table 1: Summary Table of Clinical Data for Based&g Accuracy 1 i -_--- ---- _- -_-_- -- ---I_-- Page 5 of 7 HIV-1 7.
Conclusions Drawn from the Clinical and Non-Clinical Studies I -T-----.