Ribosomal RNA
The ribosomal ribonucleic acid, abbreviated rRNA is the RNA, from which, together with about 50 protein molecules in prokaryotes and 80 eukaryotes ribosomes are constructed. It is usually at three or four different RNA molecules with Pro or eukaryotes. With the ribosomal proteins linked to the ribosome synthesized rRNA all proteins required by the cell and is thus essential for every living thing. She has in this composite enzymatic, structural and recognition functions. Thus, for example, the peptide bond of successive amino acids from the rRNA catalyzes while enzymatic activity is usually carried out by other proteins. DNA encoding the rRNA genes is referred to as rDNA. In order to meet the high demand of the cell to rRNA can - the rRNA can account for up to 70 % of the total RNA of a cell - we find the rDNA several times to many on the chromosomes.
- 2.1 rRNA as molecular markers
- 2.2 units of information
- 2.3 Selection of suitable rRNA molecules
- 2.4 Selection of appropriate sequence segments
- 2.5 Sequence Analysis
- 2.6 applications and insights
Survey
Ribosomes contain three different sizes of prokaryotic rRNA molecules, the four of eukaryotes. The size of the rRNAs is usually expressed by their sedimentation in Svedberg (S). The number of nucleotide bases or nucleotides of the rRNA molecules in question varies depending on the species. The data in the table are examples so far:
RRNA of prokaryotes
16S rRNA turns along with various proteins for about 2 /3 of the mass of the small 30S subunit of the prokaryotic ribosome, and has an important function in the initiation phase of translation: by base pairing the 3'-end of the 16S rRNA to bind to the Shine- Dalgarno sequence of the mRNA. Wherein the start codon of the mRNA is brought into the correct position in the ribosome. This position is called the P site.
RRNA of eukaryotes
Speaking of eukaryotic rRNA, the mitochondrial and plastid is always excluded. Namely, these have their own ribosomes, but are more like prokaryotic pattern. There are however many variations. So, for example, contain the mitochondrial ribosomes of many species, including the human, only two rRNA molecules.
The regions of the chromosomes harboring the rDNA accumulate in the nucleus during interphase of the cell cycle to one or more nucleoli ( nucleolus, plural nucleoli ) together. RDNA there is transcribed by RNA polymerase I, while for the production of mRNA the RNA polymerase II is responsible. At first, a pre - 45S rRNA is generated, the processing returns the 18S, 5.8S and 28S rRNAs in the same number. Only the 5S rRNA is transcribed independently thereof in another place and through the RNA polymerase III. These and the ribosomal proteins ( synthesized in the cytosol ) are brought to the nucleolus, where from both the large and small subunit of the ribosome forming in order to be then removed from the core. Naked, that is, not protected by proteins RNA would become damaged quickly. A special regulatory mechanism ensures that the 5S rRNA is formed in the appropriate amount ..
RRNA and phylogenetics
Ribosomal RNA gained enormous importance as a tool for elucidating the phylogeny, evolution of life and the exploration of relationships among organisms in recent decades. The analysis of rRNA is now an accepted method for classification of a species in the universal tree of life and to identify the most closely related species. Thus, the mentioned similarity of RNA from mitochondria and plastids strong support of Endosymbiontenhypothese for these organelles.
RRNA as a molecular marker
Ribosomal RNA was probably already part of the first living entities on earth and thus the ancestor of all organisms living today. It belongs to the basic equipment of every living cell today. At the same time she has in all organisms the same function and the genes of the rRNA probably rarely subject to horizontal gene transfer. Therefore, one assumes that the rRNA molecules evolve in all organisms with comparable speed and reflect not only the evolution of the respective rRNA gene but of a whole organism. They are considered ideal "molecular chronometer ", with the help of which have family connections among all organisms reconstruct.
However, RNA is an unstable molecule and their analysis technically complex. DNA is more stable than RNA, and easier to handle. Therefore, it is almost always in the practice of the rRNA genes, that is, the rDNA sequence thereof and runs from the rRNA.
Information units
Ribosomal RNA is - like any RNA and DNA and - of a chain of consecutively arranged nucleotides. The sequence of these nucleotides - read in a fixed direction - gives the nucleotide sequence of the rRNA molecule. This sequence contains the actual phylogenetic information. Every single base serves as a unit of information. Information about family relationships provide the differences when comparing two rRNA sequences.
Selection of appropriate rRNA molecules
The small 5 - and 5.8S rRNA due to their small number of nucleotides provide too little phylogenetic information, the analysis of the large rRNA is difficult. That is why work predominantly with 16S rRNAs in prokaryotes and 18S rRNAs in eukaryotes. This sequence databases are currently the most extensive.
Selection of appropriate sequence segments
RRNA molecules have portions that are more or less conserved. Highly conserved regions in their structure are essential for the function of ribosomes. One finds, therefore nearly unchanged in all organisms. Such sequence segments often form double-stranded secondary structures with ring-shaped loops. Changes in the base sequence are for the affected organism usually harmful or fatal. Accordingly, such mutations in a population are rarely fixed.
The base sequence of the other portions is variable. Mutations can be compensated for example by changes in ribosomal proteins here. Over time, such mutations in a population or of a species can enforce.
Finally, portions of the sequence of which can be varied almost at will without adversely affecting the operation of the ribosomes exist. These regions are hardly subject to a selection and change by a factor of thousand times faster than conserved sections.
The basic assumption for the analysis kinship relationships is that the number of fixed mutations, ie changes in the base sequence, with the time increases proportionally. To distinguish closely related organisms highly variable regions of the rRNA are, because only here you will find informative sequence differences. Distantly related or unknown organisms, however, are analyzed on the basis of highly conserved sequence segments or more.
Sequence analysis
If two species very closely related, so they have taken phylogenetically only recently developed independently. In this short time, hardly any changes in the rRNA sequences of both species have occurred. The sequences can be aligned with each other ( alignment ), they are virtually identical. Individual differences in sequence can then be relatively easily recognized.
Example:
However, comparing the rRNAs of two distantly related species, the alignment is difficult. The sequences are too different to be able to align readily to each other can. Only the highly conserved functional sections provide more information, as the sequences match. Because also vary conserved regions in their sequence, often helps a prediction of possible secondary structures ( loops ), - are functionally conserved - as indicated above.
The correct alignment of the sequences is the most important and often the most difficult part of the sequence analysis. It is necessary to compare the sequence sections of a kind with precisely the corresponding portions of the other type. ( If both sequences against each other, the result of the analysis is falsified. This is relatively easy in rRNAs of unknown species, since rRNA molecules of different species can have different lengths and whole sections are missing. )
Using mathematical method, which also reversions, so consider back mutations, a calculated " phylogenetic distance" from the recognized sequence differences, and this usually presented in the form of a Dendrogrammes or " family tree ". The greater the differences in sequence, the greater the phylogenetic distance, and the longer the " branches " in the dendrogram.
Applications and insights
The developed on the basis of ribosomal RNA pedigrees are now considered reliable and most of the hereby calculated relationships were also confirmed by other methods. Nevertheless, the application of the rRNA method can not be used alone for the correct classification of an organism. The calculated position in the pedigree must always be confirmed by other methods. To this end, still include morphological and physiological characteristics. It is for example not possible to define solely on the basis of a rRNA analysis, a new type.
Great importance has the rRNA -based phylogeny in microorganisms, because protozoa are only difficult to classify based on morphological and physiological characteristics. Here, the analysis of ribosomal RNA, a fast and reliable supplement. Based on empirical data, it is now believed that bacteria whose 16S rRNA sequences to 97-98 % match may be attributed to a species.
It has been isolated from various environmental samples (eg, water, soil and sewage sludge ) DNA and determined therefrom rRNA sequences. In one gram of forest soil were found such as rRNA genes of about 13,000 (!) Different " types ". Comparing these sequences with those of culturable and therefore known microorganisms, one can estimate that we will ever know only 1-5 % of all micro-organisms today. Of the existence of the vast majority of all bacteria and archaea is known only by their rRNA sequences without a good idea of what they live and what role they play in nature.
All previously described valid (ie, culturable ) bacteria are - depending on the author - currently classified into 26 phyla or tribes. However, the vast majority of all bacteria spread to only a few tribes, such as the Proteobacteria, Firmicutes and Actinobacteria. Most phyla are, however, only one or a few cultivable representatives represented (for example, Acidobacteria ), although it is known that these groups must involve much more representative.
26 other phyla are postulated only with the aid of isolated from environmental samples rRNA sequences without having been cultured and characterized a representative.
The most important consequence of the application of rRNA -based phylogeny was previously but the disposition of all organisms in the three domains of Bacteria, Archaea and eukaryotes. In addition, the endosymbiotic theory was confirmed. But the currently current scaling of the Urmünder ( Protostomia ), the most species-rich animal group in molting animals ( Ecdysozoa including insects, nematodes ) and lophotrochozoans ( Lophotrochozoa, including molluscs, annelids ) has been based primarily developed by investigations of the 18S rRNA of the ribosome.
Molecular markers
Other molecular markers are, for example,
- The elongation factor Tu ( EFTu )
- The gene for the subunit I of cytochrome c oxidase
- The gene of the cytochrome b of cytochrome c reductase
- The genes for the ATP synthase
- Heat shock proteins or
- ITS for very closely related eukaryotes or to distinguish between different strains