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Ribosome; the ultimate protein synthesizing nano machine

       Cell is a big factory where the instructions for all activities are coded in the DNA. A unit of instruction capable of directing the synthesis of a polypeptide or protein (or RNAs like rRNA, ribozymes etc) is termed as a gene. But the work force is the proteins which are coded by these DNAs, specifically genes. The most vital activity in this factory is undoubtedly protein synthesis. This job is done by an extreme nano machine called ribosome which synthesise proteins using decoded mRNA information (from DNA) in the cytoplasm. This 24x7 machine is the most efficient and perfect machine designed for the purpose of protein synthesis. Its amazing structural simplicity in size (just ~23nm, 1nm=10-9 M) and complexity in its function ie; synthesizing thousands of proteins required for the cell, is not easy to comprehend. I think, I don’t have the knowledge to site any known machine in size or complexity as marvellous as ribosomes and that is it.

Ribosomes are made up of several rRNA molecules and ribosomal proteins, simply an RNA-protein complex. Ribosomal RNA accounts nearly 80% of all RNAs in a cell. A large number of RNA transcripts are continuously synthesised and transported to the cytoplasm from the nucleus for protein synthesis. To meet this high demand of protein synthesising machinery, DNA sequences coding for ribosomal RNA the makes up ribosome are normally repeated hundreds of times.
Ribosome

Zamecnik discovered beyond doubt that the cellular machines responsible for protein synthesis are ribosomes. This discovery fuelled the search for different aspects of ribosomes. Later Masayasu Nomura (1960) succeeded in breaking 70S ribosome into its protein and RNA components and could spontaneously reassemble in vitro to 30S and 50S subunits under appropriate condition. These reassembled subunits resemble the native subunit in its activity and structure. High resolution structures of bacterial ribosomal subunits revealed the exact nature of RNA and protein components in the ribosomes. In 50S subunit, the 5S and 23S rRNAs form the structural core. Proteins are secondary components in the complex as there is no protein within 18A0 of the active site for peptide bond formation. As expected the RNA component in the ribosome has catalytic activity and ribosome is a ribozyme.

Each E.coli cells contain ~15000, ribosomes, making up 25% of the dry weight of the cell. In prokaryotes, ribosomes are smaller and made up of two unequal subunits 30S and 50S (Svedberg unit) and combined to form a sedimentation co-efficient of 70S (18nm diameter). Whereas,  Eukaryotic ribosomes are bigger (23nm) and more complex, 80S and made up of subunits 40S and 60S.

Eukaryotic Mammalian Ribosome components and Subunits  (Fig: 2  



Eukaryotic cells contain millions of ribosomes. Eukaryotic ribosome possess four distinct ribosomal RNAs, 3 in the large subunit 60S and one in the small sub unit 40S. In humans, the large subunit contains 28S, 5.8S and 5S RNA molecule, and the small subunit contains an 18S RNA molecule. The 28S, 18S and 5.8S rRNA molecules are first formed as a primary transcript called pre-rRNA later trimmed by nucleases to from the final product. Remember, 28S, 18S and 5.8S rRNA molecules are transcribed by RNA polymerase I located in the nucleolus whereas 5S rRNA is synthesized from genes outside the nucleolus and the enzyme involved is RNA polymerase III. The 5S rRNA after synthesis is transported to the nucleolus where it joins other components to form ribosomal subunits (Fig: 2).

 The genes coding for ribosomal RNA (rRNA) are called rDNA genes. rDNA genes are seen as clustered at specific regions. In humans, 5 rDNA clusters are located on five different chromosomes. Think about the function of nucleolus, nucleolus is the site where ribosome subunits (40S and 60S) are synthesized. Nucleolus is nothing, but the clusters of rDNA that form conspicuous irregularly shaped organisation in the nucleus during interphase.
Synthesizing the rRNA precursor.

Majority of studies on rRNA transcript synthesis were carried out on amphibian oocyte due to large size up to 2.5 mm in diameter and large numbers of nucleoli present (~100). Electron microscopic study revealed tandem repeats of rRNA genes (rDNA) are situated along the DNA molecule. The selective amplification of rDNA is necessary for production of large number of ribosomes that are required for fertilized egg to synthesize enough proteins for embryonic development. The transcription of rDNA is mediated by RNA polymerase I (an RNA pol I for every 100 bp of DNA). The rRNA precursor is further processed by RNA and proteins to form final rRNA product. Adjacent rDNA genes are separated by spacer DNA which is not transcribed.
rRNA precursor processing:

The pre rRNA is processed by two ways: 1) methylation of nucleotide residues by methylase and 2) conversion of uridine residues to pseudouridine residues by pseudouridylase. 

a)pre-rRNA modification  b) modification by snoRNA
 (Fig: 3)


All these modifications occur after nucleotide incorporation to the nascent RNA, that is post transcriptionally. These modified residues are located at specific positions and are seen as clustered together. Only the altered nucleotides take part in the rRNA formation whereas unaltered nucleotides are discarded during processing.
The function of methylation and uridine conversion is still unclear. The possible functions may be to protect pre rRNA from enzymatic cleavage, promote rRNA folding to final 3D structure, or to promote interactions of rRNAs with other molecule.


First the formation of 45S pre rRNA


This 45S rRNA is trimmed down to the 28S, 18S and 5.8S rRNA molecules (Fig :4)


Processing of Mammalian Ribosomal RNA (Fig: 4)Circled numbers are the principal cleavage sites


Other members involved in pre rRNA processing.

 snoRNAs are small, nucleolar RNAs that are present in large numbers  seen associated with particular proteins to form particles called snoRNPs (small, nucleolar ribonucleoproteins) that assist in pre rRNA processing. snoRNAs are divided into two groups based on function 1) box C/D snoRNAs decides the ribose moieties of nucleotide residues to be methylated. 2) box H/ACA snoRNAs determine the uridine residues which is to be converted to psuedouridine. This snoRNAs contain nucleotides (10-21 nucleotides), that are complementary to sections of rRNA transcript and this region bind to form RNA-RNA duplex (Fig. 3b).
Exosomes are dozens of different exonucleases involve in RNA degradation during pre rRNA processing to final rRNA.
Many findings and explanations on ribosomes are speculations based on electronmicrograph. As we discussed in the first paragraph, we are still at the shore while explaining this wonderful nanomachines.
Glossary:
  • Sedimentation coefficient: a measure of the rate at which a molecule (protein) suspended in a colloidal solution sediments in an ultracentrifuge. It is usually expressed in Svedbergs.
  • Ribozymes: RNA with catalytic activity eg: telomerase, spliceosome, peptidyl transferase etc
  • Abzymes: antibodies with catalytic activity catalytic monoclonal antibody.
Additional points:
  • Dyskeratosis is rare fatal disease characterized by skin abnormalities, bone marrow failure and high risk of cancer. This disease is due to the mutation of the enzyme that convert uridine to pseudouridine in rRNAs.  
  • Difference between spacer DNA and introns: Introns are intra genic or DNA present within the genes that are not transcribed whereas spacer DNA are intragenic and are untranscribed regions present between the genes.

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