2.7 Transcription and RNA Maturation
2.7.1 RNA Polymerases
The DNA-dependent RNA polymerases synthesize complementary RNA on the DNA matrix by
the creation of phosphodiester bonds between
the free 3'-OH groups of the RNA and the 5'phosphates of ribonucleoside triphosphates.
Whilst in bacteria, mitochondria and chloroplasts
there is only one RNA polymerase, in the cell
nucleus there are three classes (I-III) of this
enzyme. Pol I is responsible for the formation of
the pre-rRNA, Pol II for the transcription of
protein-coding genes, and Pol III for the synthesis of the tRNA precursors, the 5S RNA and
other smaller RNAs. Pol I is localized in the nucleolus and is activated by Mg2+, whereas the other
two enzymes are found in the nucleoplasm and
require Mn2+. Pol II is inhibited by as little as
1O- 7 M a-amanitin, Pol III by at least 10-4M aaminitin, and Pol I is completely insensitive. The
little-investigated mitochondrial RNA polymerases, like those of the prokaryotes, are specifically
inhibited by rifampicin [6, 37].
In spite of different specificities, the three
classes coincide in their general functional and
structural principles; the catalysed process always
follows the order: DNA sequence recognition,
binding, initiation, elongation, termination and
release of the products. All polymerases have
molecular masses between 500 and 700 kDa and
are composed of two non-identical, large polypeptides of 120-240 kDa and several others of
mostly less than 50 kDa [37,96, 377]. Comparison of gene sequences in different eukaryotes
reveals that the large subunits of the three polymerase types are homologous. Only the large
subunit of Pol II carries a C-terminal extension
made up of repeated heptapeptides, numbering
26 in yeast, 44 in Drosophila and 52 in mammals,
with the consensus sequence YSPTSPS. In contrast to all eukaryotes, Trypanosoma brucei possesses two Pol II forms that are coded by different genes, differ in only four amino acids and
have differently constructed, non-repetitive Cterminal domains. Quite surprisingly, the biosynthesis of the variable surface glycoproteins (VSG)
of the trypanosomes is not inhibited by aamanitin; ongoing studies of the three polymerases have as yet found no explanation for this
observation [404].
2.7.2 Transcription
37
2.7.2 Transcription
The initiation and termination signals for the
three polymerase classes have different locations
and structures. Around the initiation point (') of
the Pol I-transcribed rRNA genes (rDNA) there
is a sequence that can be represented by the consensus sequence ATRT' A, where R stands for a
purine; the 5' end of the 18S rRNA gene of the
silkworm Bombyx mori lies 909 bp downstream
from the initiation point [142]. Transcription of
the rDNA extends several hundred nucleotides
beyond the 3' end of the mature 28S rRNA; e.g.
in Xenopus by 235 nt and in the mouse by 565 nt
[346]. The control region for the Pol IIItranscribed genes for the small RNAs lies in the
middle of the coding sequence. In the gene for
the 5S rRNA, for example, a control protein of
37 kDa is bound at this point and guides the polymerase to its binding site in front of the coding
sequence; sequences in front of the coding region
are also required for the transcription of the 5S
rRNAgene of Bombyx mori [464]. Other Pol IIItranscribed genes most probably have specific
protein factors that correspond to the control protein of the 5S rRNA gene. The termination signal
here consists, in the simplest case, of at least
TTTT in a GC-rich region; this is found, for
example, in the Xenopus and mammalian tRNA
genes. However, there are also Pol IIItranscribed genes without poly(T) [346].
By far the most thoroughly understood are the
transcription units of the Pol II-transcribed,
protein-coding genes and their signals for the
initiation and termination of transcription, the
formation of the 3' end of the mRNA, the excision of the introns and the splicing of the exon
ends (Fig. 2.1). The human ~-globin genes may be
considered as prototypes for Pol II-transcribed
genes; the analysis of spontaneous mutations that
lead to reduced synthesis of the ~-globin chains
(~-thalassemia)
has contributed greatly to
explaining the significance of different regions
of the gene sequence [333]. The sequence
TATAAA, the TATA box or Hogness-Goldberg
box, is found at about -30 in the 5' region
upstream of the coding sequence of all Pol IItranscribed genes. There are, however, genes in
which a typical TATA is missing, e.g. the gene for
tropomyosin I in Drosophila melanogaster [23].
In many genes, there are further, less strongly
conserved elements in the region between -130
and -50; mutation of these can influence transcription, e.g. the sequences CCAAT or GGGCG
[54]. The histone genes of the ciliate Tetrahymena
2.7.1 RNA Polymerases
The DNA-dependent RNA polymerases synthesize complementary RNA on the DNA matrix by
the creation of phosphodiester bonds between
the free 3'-OH groups of the RNA and the 5'phosphates of ribonucleoside triphosphates.
Whilst in bacteria, mitochondria and chloroplasts
there is only one RNA polymerase, in the cell
nucleus there are three classes (I-III) of this
enzyme. Pol I is responsible for the formation of
the pre-rRNA, Pol II for the transcription of
protein-coding genes, and Pol III for the synthesis of the tRNA precursors, the 5S RNA and
other smaller RNAs. Pol I is localized in the nucleolus and is activated by Mg2+, whereas the other
two enzymes are found in the nucleoplasm and
require Mn2+. Pol II is inhibited by as little as
1O- 7 M a-amanitin, Pol III by at least 10-4M aaminitin, and Pol I is completely insensitive. The
little-investigated mitochondrial RNA polymerases, like those of the prokaryotes, are specifically
inhibited by rifampicin [6, 37].
In spite of different specificities, the three
classes coincide in their general functional and
structural principles; the catalysed process always
follows the order: DNA sequence recognition,
binding, initiation, elongation, termination and
release of the products. All polymerases have
molecular masses between 500 and 700 kDa and
are composed of two non-identical, large polypeptides of 120-240 kDa and several others of
mostly less than 50 kDa [37,96, 377]. Comparison of gene sequences in different eukaryotes
reveals that the large subunits of the three polymerase types are homologous. Only the large
subunit of Pol II carries a C-terminal extension
made up of repeated heptapeptides, numbering
26 in yeast, 44 in Drosophila and 52 in mammals,
with the consensus sequence YSPTSPS. In contrast to all eukaryotes, Trypanosoma brucei possesses two Pol II forms that are coded by different genes, differ in only four amino acids and
have differently constructed, non-repetitive Cterminal domains. Quite surprisingly, the biosynthesis of the variable surface glycoproteins (VSG)
of the trypanosomes is not inhibited by aamanitin; ongoing studies of the three polymerases have as yet found no explanation for this
observation [404].
2.7.2 Transcription
37
2.7.2 Transcription
The initiation and termination signals for the
three polymerase classes have different locations
and structures. Around the initiation point (') of
the Pol I-transcribed rRNA genes (rDNA) there
is a sequence that can be represented by the consensus sequence ATRT' A, where R stands for a
purine; the 5' end of the 18S rRNA gene of the
silkworm Bombyx mori lies 909 bp downstream
from the initiation point [142]. Transcription of
the rDNA extends several hundred nucleotides
beyond the 3' end of the mature 28S rRNA; e.g.
in Xenopus by 235 nt and in the mouse by 565 nt
[346]. The control region for the Pol IIItranscribed genes for the small RNAs lies in the
middle of the coding sequence. In the gene for
the 5S rRNA, for example, a control protein of
37 kDa is bound at this point and guides the polymerase to its binding site in front of the coding
sequence; sequences in front of the coding region
are also required for the transcription of the 5S
rRNAgene of Bombyx mori [464]. Other Pol IIItranscribed genes most probably have specific
protein factors that correspond to the control protein of the 5S rRNA gene. The termination signal
here consists, in the simplest case, of at least
TTTT in a GC-rich region; this is found, for
example, in the Xenopus and mammalian tRNA
genes. However, there are also Pol IIItranscribed genes without poly(T) [346].
By far the most thoroughly understood are the
transcription units of the Pol II-transcribed,
protein-coding genes and their signals for the
initiation and termination of transcription, the
formation of the 3' end of the mRNA, the excision of the introns and the splicing of the exon
ends (Fig. 2.1). The human ~-globin genes may be
considered as prototypes for Pol II-transcribed
genes; the analysis of spontaneous mutations that
lead to reduced synthesis of the ~-globin chains
(~-thalassemia)
has contributed greatly to
explaining the significance of different regions
of the gene sequence [333]. The sequence
TATAAA, the TATA box or Hogness-Goldberg
box, is found at about -30 in the 5' region
upstream of the coding sequence of all Pol IItranscribed genes. There are, however, genes in
which a typical TATA is missing, e.g. the gene for
tropomyosin I in Drosophila melanogaster [23].
In many genes, there are further, less strongly
conserved elements in the region between -130
and -50; mutation of these can influence transcription, e.g. the sequences CCAAT or GGGCG
[54]. The histone genes of the ciliate Tetrahymena