- Ribosomal DNA (rDNA) is usually a tandemly repeated series of genes coding for a precursor to the two large rRNAs.
- The nucleolus (nucleoli) is a discrete region of the nucleus where ribosomes are produced.
- The nucleolar organizer is the region of a chromosome carrying genes coding for rRNA.
- The nontranscribed spacer is the region between transcription units in a tandem gene cluster.
- Ribosomal RNA is coded by a large number of identical genes that are tandemly repeated to form one or more clusters.
- Each rDNA cluster is organized so that transcription units giving a joint precursor to the major rRNAs alternate with nontranscribed spacers.
In the cases we have discussed so far, there are differences between the individual members of a gene cluster that allow selective pressure to act independently upon each gene. A contrast is provided by two cases of large gene clusters that contain many identical copies of the same gene or genes. Most organisms contain multiple copies of the genes for the histone proteins that are a major component of the chromosomes; and there are almost always multiple copies of the genes that code for the ribosomal RNAs. These situations pose some interesting evolutionary questions.
Ribosomal RNA is the predominant product of transcription, constituting some 80-90% of the total mass of cellular RNA in both eukaryotes and prokaryotes. The number of major rRNA genes varies from 7 in E. coli, 100-200 in lower eukaryotes, to several hundred in higher eukaryotes. The genes for the large and small rRNA (found respectively in the large and small subunits of the ribosome) usually form a tandem pair. (The sole exception is the yeast mitochondrion.)
The lack of any detectable variation in the sequences of the rRNA molecules implies that all the copies of each gene must be identical, or at least must have differences below the level of detection in rRNA (~1%). A point of major interest is what mechanism(s) are used to prevent variations from accruing in the individual sequences.
In bacteria, the multiple rRNA gene pairs are dispersed. In most eukaryotic nuclei, the rRNA genes are contained in a tandem cluster or clusters. Sometimes these regions are called rDNA. (In some cases, the proportion of rDNA in the total DNA, together with its atypical base composition, is great enough to allow its isolation as a separate fraction directly from sheared genomic DNA.) An important diagnostic feature of a tandem cluster is that it generates a circular restriction map, as shown in Figure 4.14.
Suppose that each repeat unit has 3 restriction sites. In the example shown in the figure, fragments A and B are contained entirely within a repeat unit, and fragment C contains the end of one repeat and the beginning of the next. When we map these fragments by conventional means, we find that A is next to B, which is next to C, which is next to A, generating the circular map. If the cluster is large, the internal fragments (A, B, C) will be present in much greater quantities than the terminal fragments (X, Y) which connect the cluster to adjacent DNA. In a cluster of 100 repeats, X and Y would be present at 1% of the level of A, B, C. This can make it difficult to obtain the ends of a gene cluster for mapping purposes.
The region of the nucleus where rRNA synthesis occurs has a characteristic appearance, with a core of fibrillar nature surrounded by a granular cortex. The fibrillar core is where the rRNA is transcribed from the DNA template; and the granular cortex is formed by the ribonucleoprotein particles into which the rRNA is assembled. The whole area is called the nucleolus. Its characteristic morphology is evident in Figure 4.15.
The particular chromosomal regions associated with a nucleolus are called nucleolar organizers. Each nucleolar organizer corresponds to a cluster of tandemly repeated rRNA genes on one chromosome. The concentration of the tandemly repeated rRNA genes, together with their very intensive transcription, is responsible for creating the characteristic morphology of the nucleoli.
The pair of major rRNAs is transcribed as a single precursor in both bacteria and eukaryotic nuclei. Following transcription, the precursor is cleaved to release the individual rRNA molecules. The transcription unit is shortest in bacteria and is longest in mammals (where it is known as 45S RNA, according to its rate of sedimentation). An rDNA cluster contains many transcription units, each separated from the next by a nontranscribed spacer. The alternation of transcription unit and nontranscribed spacer can be seen directly in electron micrographs. The example shown in Figure 4.16 is taken from the newt N. viridescens, in which each transcription unit is intensively expressed, so that many RNA polymerases are simultaneously engaged in transcription on one repeating unit. The polymerases are so closely packed that the RNA transcripts form a characteristic matrix displaying increasing length along the transcription unit.