The Overview of Orthogonal (Aegisafe) Ribosome

Ribosome

Ribosomes consist of two subunits that fit together and work as one to translate the mRNA into a polypeptide chain during protein synthesis. Because they are formed from two subunits of non-equal size, they are slightly longer in the axis than in diameter. Prokaryotic ribosomes are around 20 nm (200 Å) in diameter and are composed of 65% ribosomal RNA and 35% ribosomal proteins. Eukaryotic ribosomes are between 25 and 30 nm (250–300 Å) in diameter and the ratio of rRNA to protein is close to 1. Bacterial subunits consist of one or two and eukaryotic of one or three very large RNA molecules (known as ribosomal RNA or rRNA) and multiple smaller protein molecules. Crystallographic work has shown that there are no ribosomal proteins close to the reaction site for polypeptide synthesis. This suggests that the protein components of ribosomes act as a scaffold that may enhance the ability of rRNA to synthesize protein rather than directly participating in catalysis (See: Ribozyme).

Ribosomes translate polypeptide chains (e.g., proteins) from the genetic instructions held within messenger RNA, using amino acids delivered by transfer RNA (tRNA). Free ribosomes are suspended in the cytosol (the semi-fluid portion of the cytoplasm); others are bound to the rough endoplasmic reticulum, giving it the appearance of roughness and thus its name, or to the nuclear envelope. Although catalysis of the peptide bond involves the C2 hydroxyl of RNA's P-site (see Function section below) adenosine in a protein shuttle mechanism, other steps in protein synthesis (such as translocation) are caused by changes in protein conformations. Since their catalytic core is made of RNA, ribosomes are classified as "ribozymes," and it is thought that they might be remnants of the RNA world.

The unit of measurement is the Svedberg unit, a measure of the rate of sedimentation in centrifugation rather than size and accounts for why fragment names do not add up (70S is made of 50S and 30S).Prokaryotes have 70S ribosomes, each consisting of a small (30S) and a large (50S) subunit. Their small subunit has a 16S RNA subunit (consisting of 1540 nucleotides) bound to 21 proteins. The large subunit is composed of a 5S RNA subunit (120 nucleotides), a 23S RNA subunit (2900 nucleotides) and 31 proteins. Affinity label for the tRNA binding sites on the E. coli ribosome allowed the identification of A and P site proteins most likely associated with the peptidyl transferase activity; labelled proteins are L27, L14, L15, L16, L2; at least L27 is located at the donor site, as shown by E. Collatz and A.P. Czernilofsky. Additional research has demonstrated that the S1 and S21 proteins, in association with the 3'-end of 16S ribosomal RNA, are involved in the initiation of translation. Eukaryotes have 80S ribosomes, each consisting of a small (40S) and large (60S) subunit. Their 40S subunit has an 18S RNA (1900 nucleotides) and 33 proteins. The large subunit is composed of a 5S RNA (120 nucleotides), 28S RNA (4700 nucleotides), a 5.8S RNA (160 nucleotides) subunits and ~49 proteins. During 1977, Czernilofsky published research that used affinity labeling to identify tRNA-binding sites on rat liver ribosomes. Several proteins, including L32/33, L36, L21, L23, L28/29 and L13 were implicated as being at or near the peptidyl transferase center.

16s ribosome RNA

16S ribosomal RNA (or 16S rRNA) is a component of the 30S small subunit of prokaryotic ribosomes. It is approximately 1.5kb (or 1500 nucleotides) in length. The genes coding for it are referred to as 16S rDNA and are used in reconstructing phylogenies.

Multiple sequences of 16S rRNA can exist within a single bacterium. It has several functions:

• Like the large (23S) ribosomal RNA, it has a structural role, acting as a scaffold defining the positions of the rebosomal proteins.
• The 3' end contains the anti-Shine-Dalgarno sequence, which binds upstream to the AUG start codon on the mRNA. The 3'-end of 16S RNA binds to the proteins S1 and S21 known to be involved in initiation of protein synthesis; RNA-protein cross-linking by A.P. Czernilofsky et al. (FEBS Lett. Vol 58, pp 281–284, 1975).
• Interacts with 23S, aiding in the binding of the two ribosomal subunits (50S+30S)
• Stabilizes correct codon-anticodon pairing in the A site, via a hydrogen bond formation between the N1 atom of Adenine (see image of Purine chemical structure) residues 1492 and 1493 and the 2'OH group of the mRNA backbone.

For the 16s of them has a structural role, acting as a scaffold defining the positions of the ribosomal proteins. The 3' end contains the anti-Shine-Dalgarno sequence, which binds upstream to the UG start codon on the mRNA. We note that the complementary position and length can have big impact on the translational result of back gene. This feature stimulates our thinking about the blueprint of our project. That is to reform some part of 16s to create our experiment result. WE do some exact test according to the paper, at last we choose this sequence to change to make our project come true.

Orthogonal (Aegisafe) Gibson Free Energy

Orthogonal Ribosome (O-Key)

Gibson Free Energy

Orthogonality Verification Experiment

Modeling

Future