|Content of Nucleic Acid Synthesis
In spite of this similarity, however, the details are
very different in eukaryotic cells and are summarized as
The fundamental process is identical in prokaryotes and
eukaryotes, in that an RNA polymerase complex binds to
the promoter and initiates transcription at a start site downstream
to the promoter. De novo initiation of anRNAchain
occurs with a purine nucleotide and creation of a transcription
bubble with the open complex. The transcription
complex can slide back along the nascent chain and endonucleolytically
cleave off the 3´ segment, then moves
forward along the DNA template chain; termination occurs
at specific regions in the genes.
- Eukaryotic RNA polymerases contain many more
subunits, located in the different regions of the nucleus.
Pol I, specific for synthesizing rRNA, is located in the nucleolus,
a specialized structure within the nucleus, while
Pol II and Pol III are in the nucleoplasm. These enzymes
have 8–14 subunits with a total molecular mass >500 kD.
The large subunits have some sequence similarity with the
bacterial RNA polymerases. RNA polymerases of mitochondria
and chloroplasts are phylogenetically closer to
bacterial RNA polymerase, commensurate with the fact
that the target genes of these enzymes are fewer and
much smaller in organelles, which are thought to have
arisen by symbiotic acquisition of bacteria by primitive
- The promoter composition and organization of eukaryotic
polymerases are quite specific for each polymerase.
The promoters of rRNA genes contain a core and
an upstream control element which is needed for high promoter
activity. Two ancillary factors, UBFl and SLl, bind
to these sequences. Although SLl binds only after UBFl in
a cooperative fashion, SL1 is a σ-factor with four proteins
among which TBP is also required for initiation by the
other polymerases. Pol I is akin to Pol III in that it utilizes
both upstream and downstream promoters. There are two
types of internal promoters with distinct sequence boxes.
One transcription factor (TFIII B) is required for initiation
of RNA synthesis by Pol III. Other factors (TFIII A
and TFIII C) help TFIII B bind to the right location and
act as positioning factors for correct localization of Pol III
Pol II is the most versatile and widely utilized RNA
polymerase in vivo and absolutely needs auxiliary, transcription
factors (TFII) whose requirement is dependent
on the nature of promoters.
- The nature of eukaryotic promoters is quite different
from the prokaryotic promoters. In addition to the bipartite
promoter of Pol I, both Pol II and Pol III have a “TATA
box” located about 25 bp upstream of the start site in Pol II
responsive genes. The 8-bp sequence consists of only AT
base pairs and is surrounded by GC pair-rich sequences.
Interestingly, the TATA box is quite similar to the −10
sequence in E. coli promoters.
There is a second element called a CAAT box, usually
about −15 bp 5´ of the TATA box. Alternatively a GC
rich sequence is present in some promoters, often at position
−90. The consensus GC box sequence is GGGCGG,
of which multiple copies are often present and occur in
both orientations. These elements are not all present in
all promoters; it appears that they work in a “mix and
match” fashion. These boxes, and also a octamer box,
bind to specific trans-acting factors and are engaged in
multiple protein interactions among themselves as well as
with components of the RNA Pol II holoenzyme.
There is no significant homology among transcription
start sites of various genes, except for the tendency for the
first base in the transcript to be an A flanked on either side
by pyrimidines. This region is defined as the initiator.
The first step in transcriptional initiation of a TATA containing
promoter is the binding of the factor TFIID to
the region upstream of the TATA site. The TATA-binding
protein, TBP, which specifically binds to the TATA box,
is a component of the TFIID complex, along with other
proteins collectively called TAFs (TBP-associated factors).
TAFs can be variable in the TFIID complex, both
in species and amounts, and provide the promoter specificity
for initiation. Some TAFs are tissue specific. TFIID
has a molecular mass of 800 kD, containing 1 TBP and
11 TAFs. TBP acts as a positioning factor and is able to
interact with a wide variety of proteins, including Pol II
and Pol III. It binds to the minor groove of the DNA
double helix and makes contact with other factors which
mostly bind to the major groove and can make multiple
contacts. By bending the DNA at the binding site, it appears
to bring the factors and RNA
polymerase into closer
Although TBP is utilized by both Pol II and Pol III,
TFIID is the specific complex for Pol II recognition of a
promoter. Other transcription factors (e.g., TFIIA) bind
to the TFIID promoter complex and cover increasing
segments of DNA. In addition to TFIIA, these include
TFIIE, TFIIF, TFIIH, and TFIIJ. Most of the TFII factors
are released from the transcription complex before Pol II
leaves the promoter and carries out chain elongation. Interestingly,
the same general transcription factors, including
TFIID, bind to the TATA-less promoter, even though
TATA binding by TBP is not available.
- A unique difference between prokaryotic and eukaryotic
transcription is that in prokaryotes a singlemRNA
containing many genes can be transcribed from the DNA
template as a single transcription unit, coupled with their
direct translation on ribosomes into discrete
This process reflects the fact that genes which encode enzymes
in a given pathway are often clustered in an operon
and are co-ordinately regulated.
In contrast to the synthesis of polycistronic mRNA in E. coli and other bacteria, eukaryotic transcription units
usually consists of single genes. This characteristic
may also reflect uncoupled transcription and translation
in these organisms. Thus, heterogeneous nuclear RNA
(hnRNA) is synthesized in the nucleus and then transported
to the cytoplasm along with its processing into mature
mRNA including splicing, addition of poly(A) tail at
the 3´ end, and capping at the 5´ end. Subsequently, the
RNA is translated on ribosomes (endoplasmic reticulum).
Thus, synthesis and utilization of mRNA are temporally
and spatially separated.