16S ribosomal RNA

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Atomic structure of the 30S Subunit from Thermus thermophilus. Proteins are shown in blue and the single RNA strand in orange.[1]

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, due to the slow rates of evolution of this region of the gene.[2] Carl Woese and George E. Fox were two of the people who pioneered the use of 16S rRNA in phylogenies.[3]

Multiple sequences of 16S rRNA can exist within a single bacterium.[4]

Functions

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

Structure

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Universal primers

The 16S rRNA gene is used for phylogenetic studies[5] as it is highly conserved between different species of bacteria and archaea.[6] Carl Woese pioneered this use of 16S rRNA.[2] Some (hyper)thermophilic archaea (i.e. order Thermoproteales) contain 16S rRNA gene introns that are located in highly conserved regions and can impact the annealing of "universal" primers.[7] Mitochondrial and chloroplastic rRNA are also amplified.

The most common primer pair was devised by Weisburg et al.[5] 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.[8] Often 8F is used rather than 27F. The two primers are almost identical, but 27F has an M instead of a C. AGAGTTTGATCMTGGCTCAG compared with 8F.[9]

Primer name Sequence (5'-3') Reference
8F AGA GTT TGA TCC TGG CTC AG [10][11]
U1492R GGT TAC CTT GTT ACG ACT T same as above
928F TAA AAC TYA AAK GAA TTG ACG GG [12]
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 [13]
1492R CGG TTA CCT TGT TAC GAC TT as above

PCR applications

In addition to highly conserved primer binding sites, 16S rRNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful for identification of bacteria.[14][15] 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.[16] Although it was originally used to identify bacteria, 16S sequencing was subsequently found to be capable of reclassifying bacteria into completely new species,[17] or even genera.[18][19] It has also been used to describe new species that have never been successfully cultured.[20][21]

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 that collect only 16S rRNA sequences are widely used. The most frequently used databases are listed below:

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.[22]

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.[23]

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 as well as a suite of search, primer-design and alignment tools (Bacteria, Archaea and Eukarya).[24]

Greengenes

Greengenes is a quality controlled, comprehensive 16S reference database and taxonomy based on a de novo phylogeny that provides standard operational taxonomic unit sets. The official home page for the site is http://greengenes.secondgenome.com, and is licensed under the Creative Commons BY-SA 3.0 license.[25][26]

References

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External links

  1. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  2. ↑ 2.0 2.1 Lua error in package.lua at line 80: module 'strict' not found.open access publication - free to read
  3. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  4. ↑ Lua error in package.lua at line 80: module 'strict' not found.
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  6. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  7. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  8. ↑ http://www.hmpdacc.org/tools_protocols.php#sequencing
  9. ↑ Primers, 16S ribosomal DNA - François Lutzoni's Lab
  10. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  11. ↑ Universal Bacterial Identification by PCR and DNA Sequencing of 16S rRNA Gene. PCR for Clinical Microbiology, 2010, Part 3, 209-214
  12. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  13. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  14. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  15. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  16. ↑ Lua error in package.lua at line 80: module 'strict' not found.
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  18. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  19. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  20. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  21. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  22. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  23. ↑ Larsen N, Olsen GJ, Maidak BL, McCaughey MJ, Overbeek R, Macke TJ, Marsh TL, Woese CR. (1993) The ribosomal database project. Nucleic Acids Res. Jul 1;21(13):3021-3.
  24. ↑ Elmar Pruesse, Christian Quast, Katrin Knittel, Bernhard M. Fuchs, Wolfgang Ludwig, Jörg Peplies, Frank Oliver Glöckner (2007) Nucleic Acids Res. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. December; 35(21): 7188–7196.
  25. ↑ Lua error in package.lua at line 80: module 'strict' not found.
  26. ↑ Lua error in package.lua at line 80: module 'strict' not found.
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