HOW DOES EUK TRANCRIPTION DIFFER FROM BACTERIAL TRANSCRIPTION?

For all eukaryotic RNA polymerases, the factors create a structure at the
promoter to provide the target that is recognized by the enzyme. For RNA
polymerases I and III, these factors are relatively simple, but for RNA polymerase
II they form a sizeable group collectively known as the basal factors. The basal
factors join with RNA polymerase II to form a complex surrounding the startpoint,
and they determine the site of initiation. The basal factors together with RNA
polymerase constitute the basal transcription apparatus.
• RNA polymerase I synthesizes rRNA in the nucleolus.
• RNA polymerase II synthesizes mRNA in the nucleoplasm.
• RNA polymerase III synthesizes small RNAs in the nucleoplasm.
• All eukaryotic RNA polymerases have — 12 subunits and are
aggregates of >500 kD.
• Some subunits are common to all three RNA polymerases.
• The largest subunit in RNA polymerase II has a CTD (carboxyi
terminal domain) consisting of multiple repeats of a septamer.
Wild and Cramer 2012
• The RNA polymerase I promoter consists of a core promoter and an upstream control
element.
• The factor UBF1 binds to both regions and enables the factor SL1 to bind.
• SL1 includes the factor TBP that is involved in initiation by all three RNA polymerases.
• RNA polymerase binds to the UBF1-SL1 complex at the core promoter.
• For high frequency initiation, the factor UBF is required. This is a single polypeptide that
binds to a G-C-rich element in the upstream promoter element. One indication of how
UBF interacts with the corebinding factor is given by the importance of the spacing
between the upstream promoter element and the core promoter.
• RNA polymerase III has two types of promoters.
• Internal promoters have short consensus sequences located
within the transcription unit and cause initiation to occur a fixed
distance upstream.
• Upstream promoters contain three short consensus sequences
upstream of the startpoint that are bound by transcription factors.

5S rRNA

tRNA

SnRNA
• TFIIIA and TFIIIC bind to the consensus sequences and enable
TFIIIB to bind at the startpoint.
• TFIIIB has TBP as one subunit and enables RNA polymerase to
bind.
• Efficiency of transcription is much increased by the presence of the PSE and
OCT elements. The factors that bind at these elements interact
cooperatively. (The PSE element may be essential at promoters used by
RNA polymerase II, whereas it is stimulatory in promoters used by RNA
polymerase III; its name stands for proximal sequence element.)
• RNA polymerase II requires general transcription factors (called TFIIX) to initiate
transcription.
• RNA polymerase II promoters have a short conserved sequence Py2CAPy5 (the initiator
InR) at the startpoint.
• The TATA box is a common component of RNA polymerase II promoters and consists of
an A-T-rich octamer located —25 bp upstream of the startpoint.
• The DPE is a common component of RNA polymerase II promoters that do not contain a
TATA box.
• A core promoter for RNA polymerase II includes the InR and either a TATA box or a DPE.
Many promoters have a sequence called the TATA box, usually located -25 bp
upstream of the start point. It constitutes the only upstream promoter element that
has a relatively fixed location with respect to the start point. The core sequence is
TATAA, usually followed by three more A-T base pairs. The TATA box tends to be
surrounded by G-C-rich sequences, which could be a factor in its function. It is
almost identical with the -10 sequence found in bacterial promoters; in fact, it could
pass for one except for the difference in its location at -25 instead of-10.

Promoters that do not contain a TATA element are called TATA-less promoters.
Surveys of promoter sequences suggest that 50% or more of promoters may be
TATA-less. When a promoter does not contain a TATA box, it usually contains
another element, the DPE (downstream promoter element) which is located at
+28 - +32. A core promoter can consist either of a TATA box plus InR or of an InR
plus DPE.
• TBP is a component of the positioning factor that is required for
each type of RNA polymerase to bind its promoter.
• The factor for RNA polymerase II is TFMD, which consists of TBP
and 11 TAFs, with a total mass - 8 0 0 kD.

First step in complex formation at a promoter containing a TATA box is binding of the
factor TFIID to a region that extends upstream from the TATA sequence. TFIID
contains two types of component.
Recognition of the TATA box is conferred by the TATA-binding protein (TBP), a small
protein of ~30 kD. The other subunits are called TAFs (for TBP-associated factors).
Some TAFs are stoichiometric with TBP; others are present in lesser amounts. TFIIDs
containing different
TAFs could recognize different promoters. Some (substoichiometric) TAFs are tissue-
specific. The total mass of TFIID typically is ~800 kD, containing TBP and 11 TAFs,
varying in mass from 30-250 kD.

TFIID is ubiquitous, but not unique. All multicellular eukaryotes also express an alternative
complex, which has TLF (TBP like factor) instead of TBP. A TLF is typically -60% similar to TBP.
It probably initiates complex formation by the usual set of TFU factors. However, TLF
does not bind to the TATA box, and we do not yet know how it works.
Drosophila also has a third factor, TRF1, which behaves in the same way as TBP and binds its
own set of TAFs, to form a complex that functions as an alternative to TFIID at a specific set
of promoters.
it surrounds one face of DNA, forming a
"saddle" around the double helix. In
effect, the inner surface of TBP binds to
DNA, and the larger outer surface is
available to extend contacts to other
proteins. The DNA-binding site
consists of a C-terminal domain that is
conserved between species,
while the variable N-terminal tail is
exposed to interact with other
proteins. It is a measure of the
conservation of mechanism in
transcriptional initiation that the DNA-
binding sequence of TBP is 80%
conserved between yeast and Man.

The TATA box bends towards the

major groove, widening the minor groove.
The distortion is restricted to
the 8 bp of the TATA box; at each end of
the sequence, the minor groove
has its usual width of ~5 A, but at the
center of the sequence the minor
groove is >9 A.
Primary
Secondary
Tertiary CAP
Importance:
1. m7G cap is required for efficient pre-mRNA splicing. This is mediated through recruiting
and binding to the nuclear cap-binding complex (CBC), which orchestrates processes such
as spliceosome assembly, 3′ processing, RNA export, miRNA biogenesis and nonsense
mediated decay. The composition and functions of CBC have been reviewed elsewhere
(39–41). Here we will focus on mRNA cap-mediated processing and nuclear export.
2. In the cytoplasm: translation initiationmRNA pseudo-circularization
The majority of cellular mRNA translation is initiated by the cap-dependent mechanism. Upon
exit into the cytoplasm, CBC stays bound to the mRNA cap and recruits eIF4G and RNA
helicase eIF4A to the 5′ end of the mRNA.
3. mRNA pseudo-circularization
When bound to the mRNA cap, eIF4G of the eIF4F complex interacts with poly(A) binding
protein PABP1 bound to the poly(A) tail of mRNA and create a pseudo-circular structure of
translating mRNA (59,60). The pseudo-circularization of mRNA has been postulated to help
ensure that full-length mRNAs are translated and enhance the processivity of the ribosome
(61,62). The m7G cap, in essence, is a unique anchor on mRNA where protein factors bind and
drive the cap-related functions for the mRNP.
4. An innate immune response built against RNA without 2′O methylation at its cap strongly
suggests that this 2′O methylation is a signature of self RNA. More detailed reviews of the
biological roles of IFIT proteins, RIG-I and MDA5
Cap snatching

In lieu of a capping machinery, some RNA viruses steal the cap from the host RNA in
a process called cap snatching. In Influenza virus ((−)ssRNA), the RNA-dependent
RNA polymerase (RdRp) is a complex of three proteins: polymerase base protein 1
(PB1), polymerase base protein 2 (PB2) and polymerase acidic protein (PA).Upon
assembly in the nucleus, the PB2 subunit binds to the cap structure of host capped
RNA. The endonuclease activity of PA then cleaves the first 10–15 nt of the capped
RNA, which is then used to prime viral mRNA transcription= Cap snatching was first
demonstrated using human globin mRNA and had since been presumed to prefer
mRNA and pre-mRNA as snatching targets. This presumption has recently been
overturned by a study that focuses on capped RNA instead of poly(A) RNA. The
study showed that that Influenza A virus prefers non-coding snRNAs U1 and U2 to
mRNA or pre-mRNA for snatching.
CAP-snatching: Pseudo circularization:
Cleavage and Polyadenylation of Pre-mRNAs
Are Tightly Coupled
In eukaryotic cells, all mRNAs, except histone mRNAs, have a 3 poly(A) tail. Early studies
of pulse-labeled adenovirus and SV40 RNA demonstrated that the viral primary transcripts
extend beyond the site in the viral mRNAs from which the poly(A) tail extends. These results
suggested that A residues are added to a 3 hydroxyl generated by endonucleolytic cleavage of
a longer transcript, but the predicted
downstream RNA fragments never were detected in vivo, presumably because of their rapid
degradation. That cleavage of a primary transcript precedes its polyadenylation was firmly
established by detection of both predicted cleavage products in in vitro processing reactions
performed with nuclear extracts of HeLa cells. Early sequencing of cDNA clones from
animal cells showed that nearly all mRNAs contain the sequence AAUAAA 10–35
nucleotides upstream from the poly(A) tail. Polyadenylation of RNA transcripts is virtually
eliminated when the corresponding sequence in the template DNA is mutated to any other
sequence except one encoding a closely related sequence (AUUAAA). The unprocessed
RNA transcripts produced from such mutant templates do not accumulate in nuclei, but are
rapidly degraded. Further mutagenesis studies revealed that a second signal downstream
from the cleavage site is required for efficient cleavage and polyadenylation of most pre-
mRNAs in animal cells. This downstream signal is not a specific sequence but rather a GU-
rich or simply a U-rich region within ≈50 nucleotides of the cleavage site. Identification and
purification of the proteins required for cleavage and polyadenylation of pre-mRNA have
led to the model shown in Figure 12-4.
• Poly(A) tail protects the mRNA molecule from enzymatic degradation in
the cytoplasm and aids in transcription termination, export of the mRNA
from the nucleus, and translation.

• Many eukaryotic non-coding RNAs are always polyadenylated at the end of

transcription. There are small RNAs where the poly(A) tail is seen only in
intermediary forms and not in the mature RNA as the ends are removed
during.

• Long noncoding RNAs (lncRNA)– a seemingly large group of regulatory RNAs

that, for example, includes the RNA Xist, which mediates X chromosome
inactivation – a poly(A) tail is part of the mature RNA.
TORPEDO MODEL
cotranscriptional
cleavage event (CoTC)