genes and many small nucleoprotein RNA (snRNA) genes. Finally, RNA polymerase IIItranscribes the
rest of the small RNAs, particularly tRNAs and 5S RNAs.
The complete nuclear RNA polymerase enzymes are multi-subunit structures of around 5 105 Da in
mass. Each polymerase is composed of two major subunits, usually about 2 105 and 1.4 105 Da,
respectively. These correspond to the and subunits of E. coliRNA polymerase. Additionally, eukary-
otic RNA polymerases contain up to ten smaller subunits of between 1 104 Da and 9 104 Da. Several
of these subunits are shared between different types of RNA polymerase.
6.6.3.2 Transcriptional Initiation for RNA Polymerase II. RNA polymerase IIis the most inter-
esting and important of the three nuclear RNA polymerases, since it transcribes all protein-encoding
genes. Initiation of transcription by RNA polymerase II is a highly complex process that is a major factor
controlling the levels of mRNAs produced and is thus key to regulating the levels of the tens of thousands
of different cellular proteins. In addition to the basic transcriptional initiation machinery,^37 there are a multi-
tude of positive and negative regulators available.^38 Many features of eukaryotic promoters are shared with
prokaryotes, in particular the basic concepts of induction and repression mediated by proteins.
6.6.3.2.1 RNA Polymerase II Promoter Structure and the Basal Transcription Machinery.
As for prokaryotes, there is a conserved box at about 25 base pairs upstream from the transcriptional
initiation site. This box, the TATA box, has the consensus TATAAATA. Deletion of this region in some
cases damages promoter strength, for example in the case of the globin promoter or many yeast pro-
moters. In other cases, TATA box removal does not abolish transcriptional initiation but destroys its speci-
ficity, leading to multiple staggered transcriptional initiation sites. Thus, the TATA box has different
functions in different genes.
The TATA box binds a protein complex called transcription factor IID(TFIID).^37 This is a multimeric
protein, one constituent of which is TATA binding protein(TBP).^39 TBP is also a constituent of transcription
factors for RNA polymerases I and III, despite the fact that these act on promoters that lack TATA boxes.
The initial steps of transcription complex assembly do not involve the polymerase at all. Instead, TFIID
binds first to the TATA box followed by two other factors, before the RNA polymerase enters the complex
(Figure 6.19). Then several other factors bind to assemble the basal transcription machinery and, finally to
complete transcriptional initiation, the carboxy-terminal domain of the 2 105 Da RNA polymerase sub-
unit is phosphorylated.
6.6.3.2.2 Regulatory Proteins Affecting RNA Polymerase II Transcriptional Initiation in
Eukaryotes. A large number of transcription factors affect transcriptional initiation.^38 For example,
transcription factorgenes constitute at least 10% of the total gene number in the genomes of Arabidopsis
thaliana and Homo sapiens. Consequently, it is not surprising that there are no consensus boxes common
to all protein-coding genes. Instead boxes are often specific to a particular class of genes that are tran-
scribed under similar conditions, in an analogous way to that described for E. coli(Table 6.2). For example,
the seven heat shock genes of Drosophilaare induced by elevated temperature. All of these genes share a
region of homology approximately 70 bp upstream of the transcription start site (the lower case letters are
less well conserved):
C T g G A A t N T T C t A G aIf several copies of this box are inserted next to a gene lacking a promoter heat inducible transcription
is observed. But instead if a mutated version of the box is inserted, transcription is abolished. Therefore,
the ‘heat shock box’ is necessary and sufficient to confer heat inducibility to a gene. The heat shock response
in eukaryotes is mediated by a transcription factor called heat shock activator protein (HAP). This protein
is always present in cell nuclei but does not induce heat shock gene transcription at ambient temperature.
Under these conditions, RNA polymerase II can bind to a heat shock promoter but stutters and only makes
short RNA transcripts, in a rather similar way to that described above for E. coli(Figure 6.17). On heat shock,
HAP forms a trimer and binds to the heat shock boxes, leading to successful transcriptional initiation.
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