rRNA, or ribosomal RNA, are molecules found in the cells which are 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

What is 16S rRNA?

Features

16s rRNA Function

Gene Detection - 16S rRNA

16S rRNA Sequence Analysis

16S Ribosomal RNA – Applications in Microbiology

rRNA

The nucleolus is a dense region within the nucleus containing genes that encode the rRNA. These rRNAs can be either small or large in size, and each ribosome must contain one of each. The ribosomal proteins are synthesised in the cytoplasm and then transported to the nucleus, where they are partially assembled in the nucleolus. Once the small and large subunits of the ribosome (50S and 30S units in bacteria, where S stands for Svedberg units) are formed, they are returned to the cytoplasm to be finished.

The RNAs found in Archaea and Bacteria differ, which is essential to know as the archaeal and bacterial lines appear to have diverged from the shared ancestor slightly before the evolution of 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 bacteria, with an associated form present in all cells, even in eukaryotes.

Research of 16S rRNA sequences from multiple organisms has revealed that certain sections of the molecule experience rapid genetic changes, which can be used to differentiate between different species within the same genus.

16S rRNA is a type of ribosomal RNA found in bacteria that is used to identify and classify different species of bacteria.

The 16S rRNA is a type of rRNA found in the small subunit of the prokaryotic ribosome, which is part of the 30S subunit. It has a structural function similar to the rRNA in the larger subunit, helping to keep the proteins of the ribosomes in place. Additionally, 16S rRNA is involved in bringing the small subunit and the large subunit together by interacting with the 23s rRNA in the larger subunit. 16S rRNA is found in the small ribosomal subunits of Archaea, Bacteria, Chloroplasts, and Bacteria.

The “S” in 16S stands for the sedimentation coefficient - an index that indicates the macromolecule’s downward velocity in a centrifugal field. The 16S rRNA gene is the sequence of DNA that corresponds to the ribosomal RNA encoding bacteria seen in the genome of bacteria. This gene is specific and highly conserved, and its sequence is quite long.

In prokaryotes, the ribosomal RNAs are:

| 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

1. 16S rRNA is a single-stranded molecule
2. It is approximately 1500 nucleotides long
3. It is highly conserved among different species
4. It is used in phylogenetic analysis to identify microbial species

Number of Copies

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

Size:

The size of the 16S rRNA coding gene is close to 1,500 bp and contains 50 functional domains.

Information

The internal structure of 16S rRNA gene consists of conserved and variable regions. Universal primers of different bacteria can be designed based on the conserved region, while particular primers of a particular bacteria can be designed based on the variable region. The interspecific variation of information in the different areas of the 16S rRNA enables specific recognition.

16s rRNA Function

The Following are the Functionalities of the 16s rRNA:

They interact with the 23S, aiding in the merging of the two ribosomal subunits (50S + 30S).

The 3’ end of the 16S rRNA has been observed to comprise a reverse SD sequence, which is used for binding the AUG codon (initiation) of the mRNA. Additionally, the combination of S1 and S21 at the 3’ terminal of the 16S rRNA has been seen to be associated with the initiation of protein synthesis.

Immobilisation of ribosomal proteins serves as scaffoldings, thus providing a structural role in defining the positions of the ribosomal proteins.

It stabilizes the precise 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 mRNA backbone.

Gene Detection using 16S rRNA

The 16S rRNA gene detection technique has become the most widely used tool to identify and detect pathogens due to the PCR technology and sustained advancements in nucleic acid research technology.

The technology is capable of quickly and accurately recognizing, categorizing, and locating pathogens. This process involves gathering genomic DNA, collecting 16S rRNA gene fragments, and analyzing the gene sequence of 16S rRNA.

16s rRNA Sequence Analysis

The 16S rRNA analysis technique involves obtaining the sequence information of 16S rRNA from its gene fragment in a microbial sample through cloning. This is followed by sequencing or probe hybridization and enzyme cutting, and then comparing the data of sequence or related information from the 16S rRNA information. This is done to place it in the evolutionary tree, thus identifying the probable samples.

The 16S ribosomal RNA sequencing is widely utilized in microbiology to identify diversity in prokaryotic and other organisms, and thus analyze the phylogenetic relationship between them.

Some benefits of using ribosomal RNA in molecular techniques are:

All cells contain ribosomal RNA and ribosomes.

RNA genes are conserved

The sequencing techniques do not involve culturing of microbial cells.

rRNA Gene Sequencing

The following steps are involved in rRNA gene sequencing:

DNA Isolation

Heating to denature the strands and using 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 product of the correct size

Purification and Sequencing of PCR Product

Related: DNA polymerase

Types of 16S rRNA Sequence Analysis

The following types can be used to analyse the 16S rRNA gene fragment:

Hybridizing the PCR products with specific probes of 16S rRNA can provide microbial constitution information. Additionally, the probe can be detected directly through in situ hybridization with the sample being tested.

The sequence of the PCR products present on the plasmid vector is compared to the sequence in the 16S rRNA database to determine its place in the evolutionary tree and identify the probable species of microbes in the sample.

Analysis of restriction fragment length polymorphism (RFLP) of PCR products was conducted. The ribosomal gene of the microbe was identified by examining the enzyme cut electrophoresis atlas, followed by numerical analysis and comparison with the data in the ribosome database. The correlation between the microbial composition of the samples and the species of various microbes was then assessed.

“16S Ribosomal RNA: Applications in Microbiology”

The 16S rRNA gene sequencing is considered to be the standard method for identifying and taxonomically classifying bacterial species.

Sequencing techniques can be utilized to describe new species which have not been successfully cultured in laboratories.

Can bacteria be reclassified into whole new genera or species?

It’s sequencing in microbiology acts as an economical and swift alternative to the phenotypic methods of identifying bacteria.

A powerful genetic method that can result in the identification of novel pathogens

Some regions of these gene sequences produce a species-specific signature sequence which can be used to identify bacteria.

Nucleotide probes are used to conduct sequence analysis, phylogenetic analysis, identify clinical bacteria, and classify bacteria molecules.