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Ribosome profiling, or Ribo-Seq (also named ribosome footprinting), is an adaptation of a technique developed by Joan Steitz and Marilyn Kozak almost 50 years ago that Nicholas Ingolia and Jonathan Weissman adapted to work with next generation sequencing that uses specialized messenger RNA sequencing to determine which mRNAs are being actively translated.
This allows separation of the Ribosome-nascent chain complex(RNC) from free mRNAs and other cell components. The RNCs form a pellet in the centrifugation that is collected for further analysis. The mRNA being translated in these RNCs can be sequenced, allowing identification and quantification of the mRNAs being translated at the time.
The ribosome has two binding sites for tRNA. They are the aminoacyl site (abbreviated A), and the peptidyl site/ exit site (abbreviated P/E). Concerning the mRNA, the three sites are oriented 5' to 3' E-P-A, because ribosomes move toward the 3' end of mRNA. The A-site binds the incoming tRNA with the complementary codon on the mRNA.
Mature mRNA is then read by the ribosome, and the ribosome creates the protein utilizing amino acids carried by transfer RNA (tRNA). This process is known as translation . All of these processes form part of the central dogma of molecular biology , which describes the flow of genetic information in a biological system.
When a ribosome finishes reading an mRNA molecule, the two subunits separate and are usually broken up but can be reused. Ribosomes are a kind of enzyme , called ribozymes because the catalytic peptidyl transferase activity that links amino acids together is performed by the ribosomal RNA.
Several ribosomes synthesizing a polypeptide on the same mRNA strand. A polyribosome (or polysome or ergosome) is a group of ribosomes bound to an mRNA molecule like “beads” on a “thread”. [1] It consists of a complex of an mRNA molecule and two or more ribosomes that act to translate mRNA instructions into polypeptides.
The ribosome catalyzes ester-amide exchange, transferring the C-terminus of a nascent peptide from a tRNA to the amine of an amino acid. These processes are able to occur due to sites within the ribosome in which these molecules can bind, formed by the rRNA stem-loops. A ribosome has three of these binding sites called the A, P and E sites:
Thus translation and transcription are parallel processes. Bacterial mRNA are usually polycistronic and contain multiple ribosome binding sites. Translation initiation is the most highly regulated step of protein synthesis in prokaryotes. [5] The rate of translation depends on two factors: the rate at which a ribosome is recruited to the RBS