section 25.5
Transcription in Eukaryotes
569
F I G U R E 2 5 - 7
Sequences found in and near some RNA polymerase II promoters. Only the TATA box is represented in many promoters
(some promotors lack this sequence). The CAAT box occurs much less frequently, and the GC box has only
occasionally been observed. Upstream sites are very common but are not considered to be part of the promoter.
RNA molecules destined to become mRNA. Polymerase
III synthesizes 5S rRNA and the tRNAs.
Polymerases II and III are inhibited by a-amanitin,
the toxic product of
Amanita phalloides
mushrooms, and
are identified by their sensitivity to this substance. Ri-
fampin, a powerful inhibitor of bacterial RNA polymerase,
is inactive against the eukaryotic nuclear polymerases
but inhibits mitochondrial RNA polymerases, although
at higher concentrations than needed to inhibit bacterial
polymerases. Thus, rifampin is used with other drugs (e.g.,
isoniazid) in treating tuberculosis. Actinomycin D is a
general inhibitor of eukaryotic transcription by virtue of
its binding to DNA. It has been useful in the treatment
of childhood neoplasms (e.g., Wilms’ tumor) and chorio-
carcinoma. However, it inhibits rapidly proliferating cells
of both normal and neoplastic origin and hence produces
toxic side effects.
RNA Polymerase II Promoters
The structure of eukaryotic promoters is more complex
than that of prokaryotic promoters. DNA sequences, hun-
dreds of base pairs (bp) upstream from the transcription
start site, control the rate of initiation. Furthermore, ini-
tiation requires numerous specific proteins (transcription
factors) that bind to particular DNA sequences. Without
the transcription factors, RNA polymerase II cannot bind
to a promoter. However, KNH polymerase II itself is not a
transcription factor. The complexity of initiation may de-
rive, in part, from the fact that eukaryotic DNA is in the
form of chromatin, which is inaccessible to RNA poly-
merases. Many RNA polymerase II promoters have the
following features:
1. A sequence, TATAAAT, about 25 bp upstream from
the transcription start site, known as the
TATA
or
Hogness box.
(Note its similarity to the Pribnow
box.) The TATA box probably determines the base
that is first transcribed.
2. A common sequence in the -75 region,
GG(T/C)CAATCT, in which T and C appear with
equal frequency at the third position. This sequence is
called the CAAT box. A third element, recognized ina
few promoters, is the GC box, GGGCGG
(Figure 25-7).
Note: The term “upstream” refers to regions in the DNA
that are to the left (or 5') of the start of transcription of
a gene; the term “downstream” refers to regions to the
right (or 3') of the gene. Regulatory regions affecting gene
expression may be located close to the transcriptional start
site, thousands of bases or more upstream or downstream
from the gene, or in introns.
RNA Polymerase III Promoters
RNA polymerase III promoters differ significantly from
RNA polymerase II promoters in that they are located
downstream from the transcription start site and within the
transcribed segment of the DNA. For example, in the 5S
RNA gene of the South African toad
(Xenopus laevis)
the
promoter is between 45 and 95 nucleotides downstream
from the start point. Thus, the binding sites on RNA poly-
merase III are reversed with respect to the transcription
direction, as compared with RNA polymerase II. That is,
RNA polymerase II reaches forward to find the start point,
and RNA polymerase III reaches backward. In fact, RNA
polymerases can slide in either direction along a DNA tem-
plate; however, they can only synthesize RNA molecules
in a
5f
—^ 3' direction.
Eukaryotic mRNA Synthesis
Eukaryotic mRNA used in protein synthesis is usually
about one-tenth the size of the primary transcript. This
size reduction results from excision of noncoding se-
quences cal 1
ed
intervening sequences
or
introns.
After ex-
cision, the coding fragments are rejoined by RNA splicing
enzymes (Figure 25-8).
Capping and Polyadenylylation
Capping occurs shortly after initiation of synthesis of
the mRNA and precedes other modifications that protect
the mRNA from degradation by nucleases. The poly (A)
