16S ribosomal RNA
16S ribosomal RNA (or 16S rRNA) is a component of the 30S small subunit of prokaryotic ribosomes. The genes coding for it are referred to as 16S rDNA and are used in reconstructing phylogenies, thanks to the work of Carl Woese and George E. Fox.
It has several functions:
- Like the large (23S) ribosomal RNA, it 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 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
The 16S rRNA gene is used for phylogenetic studies as it is highly conserved between different species of bacteria and archaea. Carl Woese pioneered this use of 16S rRNA. In addition to these, mitochondrial and chloroplastic rRNA are also amplified.
The most common primer pair was devised by Weisburg et al. and is currently referred to as 27F and 1492R; however, for some applications shorter amplicons may be necessary for example for 454 sequencing with Titanium chemistry (500-ish reads are ideal) the primer pair 27F-534R covering V1 to V3. Often 8F is used rather than 27F. The two primers are almost identical, but 27F has a M (A or C) instead of a C. AGAGTTTGATCMTGGCTCAG compared with 8F.
|Primer name||Sequence (5'-3')||Reference|
|8F||AGA GTT TGA TCC TGG CTC AG|||
|U1492R||GGT TAC CTT GTT ACG ACT T||same as above|
|928F||TAA AAC TYA AAK GAA TTG ACG GG|||
|336R||ACT GCT GCS YCC CGT AGG AGT CT||as above|
|1100F||YAA CGA GCG CAA CCC|
|1100R||GGG TTG CGC TCG TTG|
|337F||GAC TCC TAC GGG AGG CWG CAG|
|907R||CCG TCA ATT CCT TTR AGT TT|
|785F||GGA TTA GAT ACC CTG GTA|
|805R||GAC TAC CAG GGT ATC TAA TC|
|533F||GTG CCA GCM GCC GCG GTA A|
|518R||GTA TTA CCG CGG CTG CTG G|
|27F||AGA GTT TGA TCM TGG CTC AG|||
|1492R||CGG TTA CCT TGT TAC GAC TT||as above|
In addition to highly conserved primer binding sites, 16S rRNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful for bacterial identification. As a result, 16S rRNA gene sequencing has become prevalent in medical microbiology as a rapid and cheap alternative to phenotypic methods of bacterial identification. Although it was originally used to identify bacteria, 16S sequencing was subsequently found to be capable of reclassifying bacteria into completely new species, or even genera. It has also been used to describe new species that have never been successfully cultured.
16S Ribosomal Databases
The 16S rRNA gene is used as the standard for classification and identification of microbes, because it is present in most microbes and shows proper changes. Type strains of 16S rRNA gene sequences for most bacteria and archaea are available on public databases such as NCBI. However, the quality of the sequences found on these databases are often not validated. Therefore, secondary databases which collect only 16S rRNA sequences are widely used. The most frequently used databases are listed below:
1) EzTaxon-e. http://eztaxon-e.ezbiocloud.net/ The EzTaxon-e database is an extension of the original EzTaxon database. It contains comprehensive 16S rRNA gene sequences of taxa with valid names as well as sequences of uncultured taxa. EzTaxon-e contains complete hierarchical taxonomic structure (from phylum rank to species rank) for the domain of bacteria and archaea.
2) Ribosomal Database Project. http://rdp.cme.msu.edu/ The Ribosomal Database Project (RDP) is a curated database that offers ribosome data along with related programs and services. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software packages for handling, analyzing and displaying alignments and trees. The data are available via ftp and electronic mail. Certain analytic services are also provided by the electronic mail server.
3) SILVA. SILVA provides comprehensive, quality checked and regularly updated datasets of aligned small (16S/18S, SSU) and large subunit (23S/28S, LSU) ribosomal RNA (rRNA) sequences for all three domains of life (Bacteria, Archaea and Eukarya).
4) Greengenes. The greengenes web application provides access to the 2011 version of the greengenes 16S rRNA gene sequence alignment for browsing, blasting, probing, and downloading. The data and tools presented by greengenes can assist the researcher in choosing phylogenetically specific probes, interpreting microarray results, and aligning/annotating novel sequences.
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- University of Washington Laboratory Medicine: Molecular Diagnosis | Bacterial Sequencing
- The Ribosomal Database Project