Ribosomal DNA
As ribosomal DNA (rDNA) those portions of the deoxyribonucleic acid (DNA ), respectively, which contain the genes for ribosomal RNA (rRNA). rRNA is a major component of ribosomes where protein synthesis (translation ) occurs. Thus, in the rRNA is a structural ribonucleic acid ( RNA) whose information is not rewritten into proteins.
In eucaryotic cells there is rDNA not only the nucleus but also in the mitochondria, and in addition in the plastids of plants. The read off of them rRNA is used only in the ribosomes of these organelles, not in the cytoplasm. Mitochondrial and plastid rDNA similar to that of their sequence of rDNA in prokaryotes, thus the endosymbiotic theory is supported.
As a eukaryotic cell requires large amounts of rRNA, its genome may have a plurality of sections, each containing many copies of genes in tandem behind the other. In humans, these sections can be found, for example, on 10 of 46 chromosomes. Here not in all cells containing chromosomal rDNA getting all sections are read and thus used for rRNA synthesis. The active rDNA sections are focused on one or a few dense regions in the nucleus, the nucleoli (also: nucleoli; singular: nucleolus ) or nucleoli. The sections containing rDNA are therefore also referred to as the " nucleolus organizing regions " ( NORs ). Prokaryotes, mitochondria and plastids do not form a nucleolus.
The special properties of these repetitive chromosome segments have to introduce a special name, just rDNA performed. Is erroneously with " rDNA " also sometimes referred to respectively, the recombinant DNA technology for producing the same. The proportion of the rDNA on the genome is different for different organisms. In humans, there are about 0.4 %, in Drosophila melanogaster is around 17 %.
- 2.1 plastid
- 2.2 mitochondria
- 3.1 18S - 5.8S, 28S rDNA cluster
- 3.2 5S rRNA genes
- 3.3 Regulation of rRNA content
- 4.1 Evolution of the rDNA
- 4.2 Evolution of the rDNA
- 7.1 prokaryotes
- 7.2 eukaryotes
RDNA in prokaryotes
Although archaea and bacteria form their own empires, their rRNA and their rDNA is organized similarly. Both types of prokaryotes typically have only three different rRNAs: 16S, 23S and 5S rRNA. Their genes are often referred to as cluster together and are transcribed together, so they form an operon.
Bacteria
The number of the rDNA operon in bacteria may vary, for example, two in Mollicutes, in Escherichia coli of seven or ten in Bacillus subtilis. The operons are usually with continuous characters (used in E. coli: rRNA to rrnG ). Often, for example in E. coli of the rDNA cluster also contains genes for tRNAs. What and whether tRNA genes are contained, is highly variable.
The primary transcript is referred to as the prokaryotic rDNA 30S pre- rRNA. The 16S and 23S rRNA are cut out by the ribonuclease III. The " fine-cut ", and the processing of the 5S rRNA and tRNA is carried out by further cutting RNA enzymes. This is not the functional rRNA areas are cut, they are methylated immediately after the transcription and thus protected.
Due to the large variety of bacteria, there are also special developments. With mycoplasma, for example, is the 5S rDNA separated form of the 16S and 23S rDNA. When Planctomycet Planctomyces limnophilus two sets of ribosomal genes are present, the 16S rDNA is separately from the clusters of closely spaced 23S and 5S rDNA.
Archaea
In some archaea, the 5S rDNA is located a little further away from the other two rDNAs. Sometimes (eg, Thermococcus celer ), there is also another, a few 1000 base pairs away 5S rDNA.
RDNA in eukaryotic organelles
The rDNAs of mitochondria and plastids are in principle prokaryotenähnlich, but have many special developments.
Plastids
The ribosome structure, the basic sequence of the rRNA genes ( 16S - 23S -5S ) and their sedimentation coefficient ( 70S ) are prokaryotenähnlich. There are also in the chloroplast tRNA genes in rDNA transcription unit. The rDNA clusters with respect to the length of the tRNA genes and also the introns within the rRNA genes exhibit significant differences. It is sometimes speculated that the chloroplast genomes of plant major groups could be of different origin.
In higher plants such as tobacco (Nicotiana tabacum ) and rice ( Oryza sativa) has been described by way of derogation from the basic structure still a 4.5S rDNA between the 5S and the 23S rDNA. This is homologous to the 3 ' end of the 23S rDNA. The 4.5 S rRNA with the 23S and 5S rRNA of the large ribosomal subunit. The green alga Chlamydomonas reinhardtii has between the 16S and 23S rDNA still a 7S and 3S- rDNA. This combination is unique in this alga.
The rDNA is often located in the so-called " inverted repeats ", two long, opposite repeating units, such as in the original Glaucocystaceae C. paradoxa, the Olisthodiscus luteus ( Raphidophyceae ) and most green plants. In many butterfly flower plants and pines of the inverted repeat is gone secondarily lost. In the red alga Porphyra purpurea and Euglena gracilis there is no inverted repeats .. In Euglena are three repeats and an additional 16S rDNA in direct repeat units together. In some Chromalveolata, such as in dinoflagellates and brown algae, as Pylaiella littoralis, there are several Plastidenchromosomenringe. In P. littoralis the rDNA is present on a ring in the inverted repeats as in green plants. In the small ring chromosome are a 16S pseudogene and away from a split 23S rDNA gene.
Mitochondria
The arrangement of the rDNA in mitochondria is very heterogeneous. It is often heavily modified from the prokaryotic form, so that at times the hypothesis was considered that the endosymbiosis of mitochondria during evolution had taken place several times.
Usually, as well as in humans, only two rRNA genes are present, one for the rRNA of the large and small subunit of the ribosome. Both genes can sometimes be removed from each other. The rRNAs formed are much smaller than those of prokaryotes: In animals 12S in the small ribosomal subunit and 16S in the large, in fungi 15S in the small and in the large 21S subunit.
A stand-alone, the 5S rDNA similar version has been described only in some green algae (eg Prototheca wickerhamii ) in land plants and in the unicellular Reclinomonas americana. Reclinomonas is considered purest mitochondria owning eukaryote. In contrast, red algae, the green plants are evolutionarily closer than this, the 5S rDNA in turn is no longer detectable.
The rDNA is only in land plants still in the form of an operon. In green algae such as Chlamydomonas reinhardtii, the rDNA genes are pieced and interrupted by other genes. Even with Reclinomonas the organization is no longer clearly as operon.
RDNA in the nucleus of eukaryotic cells
The cytoplasmic ribosomes of eukaryotes contain four different rRNAs. This can account for up to 90 % of the total RNA of a cell. For the production of such large amounts of many genes will be required for each of the four rRNAs. The genes of three (corresponding to the 18S, 5.8S and 28S rRNA) are each directly after each other and are transcribed together, from the RNA polymerase I. Such transcription units are in large numbers as a tandem repeat units (English: repeats) before they form the actual rDNA ( see figure). The microscopic image of the metaphase the repeat units can be seen as secondary constrictions ( constriction ), since the rDNA is present particularly packed.
The genes of the 5S rRNA form a separate gene family, which are not strictly part of the rDNA. They are transcribed by RNA polymerase III. In some yeasts such as Hansenula polymorpha or Saccharomyces cerevisiae, they are located directly behind the 18S, 5.8S, 28S repeats, but on the opposite strand, ie in the opposite direction of reading. For most yeast and other eukaryotes, the remaining 5S rRNA genes are off the 18S, 5.8S, 28S repeats. They also often form tandem repeats.
For the synthesis of the ribosome, the exact same number of all the rRNAs required. By the spatial separation of the 5S rRNA gene is not automatically made in eukaryotes (as opposed to the prokaryotes ) the same amount, so that a special regulation is required (see below).
The rDNA is usually part of the chromosomes. Only in some protists, such as the slime mold Physarum species it is extrachromosomal ago.
18S - 5.8S, 28S rDNA cluster
The 18S, 5.8S and 28S rRNA genes of a transcription unit are separated at the DNA level by two so-called internal transcribed spacer (ITS) and co-led by an external transcribed spacer (ETS ) (see diagram ). Sometimes there are specific on repeat end still an ETS. Successive transcription units are separated by non- transcribed spacer ( NTS).
The nichtranskribierten spacer before each repeat blocks consist of a multiplication of promoters ( total of 2300-5300 bp ) so that they have a high bonding strength for RNA polymerases. The high efficiency promoter causes the rDNA genes are transcribed simultaneously in many cases, resulting in the so-called " Miller spreading " through " tree structures " shows up ( see picture).
From the 45S pre- rRNA is separated first, the external transcribed spacer, arises the 41S rRNA precursor. This is cleaved in the first ITS in a 20S pre- rRNA and a 32S precursor. In the third step, the precursor of the small 20S ITS part is removed and the mature 18S rRNA produced. Simultaneously, the ITS from 32S precursors are excised and the 5.8S rRNA bound to the complementary central part of the 28S rRNA. These reactions are catalyzed by the so-called small nucleolar ribonucleoprotein particles ( snoRNP ).
In Drosophila but also in many other organisms, the 28S rDNA can contain a so-called intervening sequence ( IVS). This is the " group I introns " attributed to which splice autocatalytically. In this case, an external guanosine monophosphate linked to the 3'- OH of the intron and beginning of the now freed 3 'end of the first exon to the 5' end of the second exon. Wherein the intron is released, and reduced combined exons. When this takes place during splicing of the rRNA procession, however, this has not been clarified.
Depending on the species, the arrangement, distribution and the number of transcription units can vary greatly. Drosophila males have about 150 copies, the females 250 for the faba bean (Vicia faba) 22,000 copies have been described.
In humans, the approximately 43 kb 18S -5, 8S - 28S transcription units are arranged in tandem to 30 or more as a cluster. The ten clusters are located on the acrocentric chromosome pairs 13, 14, 15, 21 and 22, the total number of transcription units is estimated to be 500 copies. Each transcription unit has its own complex regulated promoter. Between the repeating units per cluster, there are periodic so-called spacer promoter in NTS areas. This transcribe a noncoding RNA that regulates the transcription of the transcription units each with a protein complex called NoRC.
5S rRNA genes
5S rRNA genes are also present in tandem repeats. In humans, a block on the long arm of chromosome 1 near the telomere is present in Drosophila melanogaster a block on chromosome 3 In the clawed frog (Xenopus laevis ) are approximately 24,000 copies near the ends of most chromosomes distributed. The DNA segments between 5S rRNA genes are also called non- transcribed spacer. You can vary greatly both between individual gene copies as well as between closely related species.
5S rRNA genes are in contrast to the other rRNA genes, but as well as the tRNA, RNA polymerase III transcription. They have no introns, and are not processed. The promoter is located within the coding sequence and internal control region is called. It consists of a box A, an intermediate element and a box C ( from 5 'to 3').
The 5S rRNA genes of the clawed frog differ within an individual by the fact that some, others are only in oocytes, however, only in somatic cells active.
Regulation of rRNA content
Eukaryotic cells must ensure that 5S RNA and other RNAs are produced in the same amounts. A different processivity of RNA polymerases I and III is balanced among other things by different copy numbers of rRNA genes. In addition, the different structure of each NTS regions lead to differential regulation. Thus, in certain situations, only a portion of the cellular rDNA is transcribed.
In some situations, however, the total content of the rDNA genes must be altered. In the polytene chromosomes of Drosophila rDNA is unterrepliziert for example. More often, however, it is necessary to produce more ribosomes, such as oocytes. This is an intracellular duplication (amplification ) of the genes allows. Thereby rDNA genes are excised from the chromosome, circularized and amplified by rolling circle replication. This dyeable strong 1901 first described DNA rings are named after their discoverer Giardina -bodies. They have been observed about the diving beetles, the crickets, the clawed frog and the hamster. The molecular mechanism is still unclear.
Ribosomal DNA evolution and
Ribosomes are complex and essential elements of each cell, accordingly, the preservation is quite high. In eukaryotes are in contrast to prokaryotes, several hundred copies, so that an independent evolution would be expected. The clusters will have homogenized by inäquales crossing over and thus inherited as a single gene. In hybrids, both of which have genomes of the progenitors ( allotetraploid ), only the rDNA of one parent is transcribed, which is called " nucleolar dominance ".
Evolution of the rDNA
Especially noticeable is the repeat structure of the rDNA. It is found in bacteria, archaea, eukaryotic cytoplasm and in some (vegetable) mitochondrial DNAs. So is quite catchy that the 16S rDNA to the 18S rDNA and 23S rDNA are homologous to the 28S rDNA. The 5.8S rDNA is homologous to the end of the 23S rDNA of prokaryotes The 5S rDNA of prokaryotes is homologous to the 5S rDNA of eukaryotes.
Such as especially the rDNA in mitochondria shows there may be various changes in the arrangement, without the functionality of the ribosomes must suffer. Since the precise interactions of the rRNAs are not yet understood enough, many questions still remain open.
Evolution with the rDNA
The importance of the rDNA is very interesting for evolutionary researchers, as defective copies of genes are predominantly harmful, and that for all protein synthesis of the cell, and are selected against. In addition, ribosomes are present in all living things, even the bridge between prokaryotes and eukaryotes is possible. For example, the 16S rDNA is used to systematize major groups of bacteria. But even with the big scheme of eukaryotes, the sequences of the mature rRNA of the ribosome subunits are quite a popular marker. The rDNA of plastids and mitochondria is also often used.
The comparison of ribosomal DNA between prokaryotes and eukaryotes, the sequences are identical, only a small extent. The folds of the rRNAs have a tremendous morphological similarity despite the different base composition.
Can not be directly influenced by selection ITS sequences play an important role in the phylogeny of the genus and species level. Usually, the 5.8S rDNA is equal mitsequenziert because they hardly falls in magnitude to the weight. To get statistically greater certainty, the ITS sequences are supplemented by at least one other marker. The use of ITS sequences has now fallen, on the one hand, as other markers of the nucleus are available and on the other hand, since the use of ITS brings greater problems for the molecular Phylogenetiker with it. Thus, although the individual bases are not conserved, but well structural elements, such as folds, so that mutations are not free. Furthermore, the alignment of different ITS copies in hybrids in just a few generations can take place, so that ITS can phylogenetically similar behavior chloroplasts. Nevertheless, the high copy number, and thus the greater availability in the laboratory is a good argument ITS continue to use.
History
Ribosomes of eukaryotes consist of four different rRNAs, each occurring at the same number. Cyrus Levinthal et al. found out in 1962 that rRNAs and tRNAs are transcribed from DNA and not be able to replicate independently. In 1965, Sol Spiegelman and Ferruccio Ritossa demonstrated by hybridization of rRNA with Drosophila cell nuclei with different numbers of Nukleolusorganisatorregionen that these regions contain the coding rDNA.
Carl Woese published in 1977 his tree of life, a tree that could bring all the major groups for the first time in context. This was based on sequences of the 16S rDNA from mitochondria and put the Archaea out as a separate kingdom.
Thomas R. Cech discovered in 1982, the intervening sequence in the 28S rRNA of the ciliate Tetrahymena thermophila, a autocatalytic splicing intron. For this groundbreaking discovery that RNA also assume enzymatic functions and can splice itself, he was with Sidney Altman 1989 Nobel Prize in Chemistry.