16S rRNA

rRNA or ribosomal RNA are molecules found in the cells involved in the protein synthesis of organelles, referred to as ribosomes that spread out to the cytoplasm. This is to aid in translating the information contained in the mRNA (messenger RNA) into the proteins. There are three important RNAs occurring in the cells – rRNA, mRNA, and tRNA (transfer RNA).

Table of Contents

rRNA

These molecules are produced in the nucleolus that seems like a dense region in the nucleus containing genes that encode the rRNA. These encoded rRNAs vary in size (small or large). At the least, each of the ribosomes comprises one large rRNA and one small rRNA. Both these in the nucleolus combine with the ribosomal proteins forming the large and small subunits of the ribosomes (in bacteria – 50S and 30S units – S stands for Svedberg units). The ribosomal proteins are synthesised in the cytoplasm and moved to the nucleus to be sub-assembled in the nucleolus. Then, the subunits are given back to the cytoplasm for concluding assembly.

The rRNAs found in Archaea and Bacteria differ. This is essential to know, as the archaeal and bacterial lines seem to have separated from the common precursor slightly prior to the development of the eukaryotic cells.

16S rRNA is a sequence of DNA encoding the RNA of the small subunit of the ribosome of bacteria. This 16S rRNA gene can be seen in all the bacteria, an associated form occurring in all the cells even in eukaryotes.

Research on 16S rRNA sequences from several entities suggests that some part of the molecule experiences speedy genetic alterations, hence differentiating between various species in the same genus.

Ribosomal RNA

What is 16S rRNA?

16S rRNA is a type of rRNA involved in making the small subunit of the prokaryotic ribosome; these are components of the 30S subunit (prokaryotic ribosome). In general, it has a structural function alike the rRNA in the larger subunit, to hold the proteins of ribosomes in set positions. They are also involved in promoting the fusion of the small subunits to the larger subunits by the interaction with the 23s rRNA in the larger subunit. The small ribosomal units in Archaea, Bacteria, Chloroplasts and Bacteria contain the 16S.

The “S” in 16S is the sedimentation coefficient – an index that indicates the macromolecule’s downward velocity in the centrifugal field. The 16S rRNA gene is the sequence of DNA that corresponds to the rRNA encoding bacteria seen in the genome of bacteria. These are specific and highly conserved; their gene sequence is lengthy enough.

In prokaryotes, the ribosomal RNAs are as follows –

Name Where can it be seen? Size
5S Large subunit of ribosome 120 nucleotides
16S Small subunit of ribosome 1500 nucleotides
23S Large subunit of ribosome 2900 nucleotides

Features of 16S rRNA

Number of copies

Bacteria comprise approximately 5 to 10 copies of the 16S rRNA, making the detection extremely sensitive.

Size

The size of the 16S rRNA coding gene is close to 1500bp containing 50 functional domains.

Information

The internal structure of 16S rRNA gene comprises conserved and variable regions. The universal primers of different bacteria could be framed as per the conservative region, and particular primers of particular bacteria could be framed as per the variable region. The interspecific variation of information in the different areas of the 16S rRNA makes the recognition specific.

16s rRNA Function

The following are the functionalities of the 16s rRNA –

  • They interact with the 23S helping in the integration of the two units of ribosomal subunits (50S+30S)
  • 3’end comprises a reverse SD sequence used in binding the AUG codon (initiation) of the mRNA. The 16S rRNA’s 3’ terminal with S1 and S21 combination was seen to be associated with the initiation of the synthesis of proteins
  • Immobilisation of ribosomal proteins serves as scaffoldings. Hence, they have a structural role in defining the positions of the ribosomal proteins
  • It stabilises the accurate codon-anticodon pairing in the A-site to form a hydrogen bond between the N1 atoms of the adenine residues, and the 2′OH group of the backbone of the mRNA

Gene detection – 16S rRNA

The technique of 16S rRNA gene detection has emerged to have become the most widely used tool to identify and detect pathogens as a result of the PCR technology, and sustained advancements in nucleic acid research technology.

The technology is applicable to recognize, categorise and find pathogens at a faster pace and with accuracy. It involves these steps – genomic DNA is gathered, collecting 16S rRNA gene fragments, and analysing the gene sequence of 16S rRNA.

16s rRNA Sequence Analysis

The fundamental principle involved in the analysis technique of 16S rRNA is in obtaining the sequence information of 16S rRNA from its fragment of the gene in the sample of the microbe through cloning. This is followed by the sequencing or probe hybridization and enzyme cutting, and then its comparison with the data of sequence or related information in the 16S rRNA information. This is carried out to find its place in the evolutionary tree, hence identifying the probable samples.

The 16S ribosomal RNA sequencing is used widely in microbiology to find diversity in prokaryotic entities and other entities, and hence study the phylogenetic association between them.

Some benefits of using ribosomal RNA in molecular techniques are –

  • Ribosomal RNA and ribosomes are seen in all the cells
  • RNA genes are conserved
  • The culturing of microbial cells is not seen in the sequencing techniques

rRNA gene sequencing

The rRNA gene sequencing involves the following steps –

  • DNA isolation
  • Heating to separate the strands and specific primers
  • Primer extension with DNA polymerase
  • Repeat the above steps to obtain multiple copies of the 16S rRNA gene
  • Run agarose gel, checking for the accurately sized product
  • Purification and sequencing of PCR product

See also: DNA polymerase

Types of 16S rRNA sequence analysis

The 16S rRNA gene fragment can be analysed in the following types –

  • Hybridization of the PCR products with the specific probes of 16S rRNA to get microbial constitution information. Additionally, the probe can be detected directly by the in situ hybridization with the testing sample
  • The PCR products present on the plasmid vector are sequenced and compared with the sequence in the 16S rRNA database to find their place in the evolutionary tree, and find the probable species of microbes in the sample
  • Analysis of restriction fragment length polymorphism of PCR products was performed. The ribose kind of microbe gene was found by noting the enzyme cut electrophoresis atlas, and then numerical analysis followed by studying with the information in the ribosome collection. The association between the microbial constitution of samples and species of various microbes was analysed

16S Ribosomal RNA – Applications in Microbiology

  • The 16S rRNA gene sequencing is considered to be the standard method to identify, and for taxonomic classification of bacterial species
  • Sequencing techniques can be utilised in describing new species, which were never cultured successfully in labs
  • Can reclassify bacteria into whole new genera or species
  • It’s sequencing in microbiology acts as an inexpensive and quick substitute to the phenotypic techniques of identifying bacteria
  • A powerful genetic method that can result in the identification of novel pathogens
  • Some regions of these gene sequences render a species-specific signature sequence used in identifying bacteria
  • The nucleotide probes are applied to identify sequence analysis, phylogenetic analysis, clinical bacteria, and bacteria’s molecular classification

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