Ribosomes are key to protein synthesis in the cell whether it be a prokaryote or eukaryote.
Ribosomes were first observed in 1953 by the Romanian cell biologist Emil Palade who relied on the electron microscope. These ribosomes appear under the microscope as small organelles. In prokaryotes, they reside freely in the cytoplasm and protoplasm of all cells. In eukaryotes they are invariably attached to the rough endoplasmic reticulum rather than being free. As a molecular structure, ribosomes are extremely large and complex.
The ribosomes are the principle site of protein synthesis. These structures ‘read’ RNA which is the blueprint for producing proteins using the process of translation. There are excellent reviews about these organelles by Green and Noller, (1997).
Structure of Ribosomes
The ribosomes are made up of proteins and RNA (ribonucleic acid) and form the basic structure of this organelle. The RNA making up the ribosome is unique and highly specific in its action. It is known as ribosomal RNA (rRNA) and is distinct from messenger RNA (mRNA) and transfer RNA (tRNA). This rRNA behaves with enzymatic properties. It is used to catalyse the formation of peptide bonds between amino acids.
The interesting feature of ribosomes in eukaryotic cells is that they are much larger than those in prokaryotes. What is common to both, is that each ribosome is composed of a small subunit and a large subunit. The other interesting feature is that ribosomes, unlike other organelles are not surrounded by any cell membrane.
The ribosomes are classified according to their sedimentation rate hence the initial S proceeding a figure. S stands for Swedeberg unit.
Prokaryotic cells contain 70S ribosomes and are composed of 30S and 50S subunits. These particular ribosomes are found freely in the cytoplasm but also in the chloroplasts and mitochondria of eukaryotic cells. They have a length of between 200 and 290 Angstroms and a diameter of between 170 and 210 Angstroms. They have a weight of 2.7 to 3.o million daltons. The rRNA to protein ratio is 60 to 40. Protein synthesis by the 70s ribosome is inhibited by antibiotics such as chloramphenicol.
Eukaryotic cells contain the 80S ribosomes which are composed of 40S and 60S subunits. These ribosomes are found freely in the cytoplasm or attached to the rough ER. They have a length of 300 to 340 Angstroms with a diameter of between 200 and 240 Angstroms. They are the heavier of the Ribosomes with a rRNA to protein ratio of 40 to 60. Protein synthesis is not inhibted by antibiotics such as chloramphenicol.
The large subunit has the specific function of being the site of translation. The rRNA along with particular proteins in this subunit take the tRNA molecules which have unique amino acids attached to them and then catalyse the condensation reactions that occur between amino acids. Hence, peptide bonds are formed between these amino acids to form a peptide chain which becomes a protein.
The mRNA which contains the code sits between the two subunits with the ribosome moving along the mRNA which is translated into a peptide/protein.
The Significance of the A and P Sites on a Ribosome
The A and P sites are specific locations on the ribosome where various components involved in protein synthesis interact. They are part of the large subunit of the ribosome and play crucial roles in the process of translation.
A site (Aminoacyl site)
The A site is where the incoming aminoacyl-tRNA (charged tRNA) binds to the ribosome during protein synthesis. It is located on the large ribosomal subunit and stands for “aminoacyl” because it accommodates the aminoacyl-tRNA carrying the next amino acid to be added to the growing polypeptide chain. The anticodon of the aminoacyl-tRNA base pairs with the complementary codon on the mRNA, ensuring accurate codon-anticodon recognition. The A site provides a docking platform for the aminoacyl-tRNA to deliver its amino acid for peptide bond formation.
P site (Peptidyl site)
The P site is where the peptidyl-tRNA is positioned on the ribosome. It is also located on the large ribosomal subunit and stands for “peptidyl” because it holds the peptidyl-tRNA, which carries the growing polypeptide chain attached to its amino acid. The peptidyl-tRNA is situated in the P site after peptide bond formation has occurred in the A site. The P site helps to maintain the correct reading frame and alignment of the mRNA on the ribosome.
During each round of translation, the ribosome advances along the mRNA in a process called translocation. This movement shifts the peptidyl-tRNA from the A site to the P site, and the uncharged tRNA from the P site to the exit site (E site). The A site is then ready to receive the next aminoacyl-tRNA, and the process of protein synthesis continues.
The A and P sites, along with the mRNA and tRNA molecules, form a functional complex within the ribosome that ensures accurate decoding of the genetic information and proper assembly of the polypeptide chain. The interactions and movements within these sites are critical for the stepwise elongation of the nascent polypeptide during translation.
The third and final binding site is the E site for t-RNA binding during translation. The E stands for exit.
How did Puromycin Play Such A Critical Role in Defining The A and P Sites?
Puromycin, an antibiotic derived from Streptomyces alboniger, has played a crucial role in elucidating the functions of the A (aminoacyl) site and P (peptidyl) site on the ribosome. It is a structural analog of the 3′-terminal end of aminoacyl-tRNA, which enables it to interact with the ribosome with subsequent incorporation into growing polypeptide chains.
Inhibition of Protein Synthesis
Puromycin acts as a potent inhibitor of protein synthesis by binding to the ribosome and mimicking the aminoacyl-tRNA molecule. It enters the A site of the ribosome, which is normally occupied by an incoming aminoacyl-tRNA during translation.
Peptide bond formation
Once puromycin is bound to the ribosome’s A site, it undergoes a reaction similar to peptide bond formation. The terminal aminoacyl group of puromycin reacts with the peptidyl-tRNA in the P site, forming a peptide bond between them.
Premature termination of translation
After the formation of the peptide bond, puromycin becomes covalently attached to the nascent polypeptide chain. This interaction prematurely terminates protein synthesis, leading to the release of the incomplete polypeptide from the ribosome.
Defining the A and P sites
The action of puromycin demonstrated that the A site is responsible for accommodating incoming aminoacyl-tRNA during protein synthesis. Puromycin’s ability to bind to the A site and mimic the role of aminoacyl-tRNA indicated that this site is involved in the recognition and selection of the correct aminoacyl-tRNA for incorporation into the growing polypeptide chain.
Ribosome translocation
In addition to defining the A site, puromycin also provided insights into the function of the P site. Once puromycin is incorporated into the nascent polypeptide chain, the ribosome is unable to translocate along the mRNA. This suggested that the P site is responsible for holding the peptidyl-tRNA, ensuring proper positioning and movement of the ribosome during translation.
Mechanistic insights
Puromycin’s action on the ribosome has helped uncover critical aspects of translation mechanisms. It highlighted the importance of accurate aminoacyl-tRNA selection in the A site and demonstrated the role of peptidyl-tRNA in peptide bond formation and ribosome translocation.
Overall, the use of puromycin in studying ribosome function and protein synthesis provided valuable evidence for the existence and roles of the A and P sites. It helped establish the A site’s involvement in aminoacyl-tRNA selection and the P site’s role in peptidyl-tRNA positioning and ribosome translocation. These findings significantly contributed to our understanding of the molecular mechanisms underlying protein synthesis.
A number of ribosomes can be attached to the same piece of mRNA to produce a polypeptide. In this way, many copies of the same protein can be produced using the same mRNA.
Conclusions
Ribosomes are extremely important to each cell because they are the sites for protein synthesis.
References
Green, R., & Noller, H. F. (1997). Ribosomes and Translation. Annual Review of Biochemistry, 66(1), pp. 679-716 (Article).
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