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1.
DNA replication and transcription are similar in a number of respects, starting with
the central fact that both involve enzyme-mediated copying of a DNA template to create a
new polynucleotide. Also, both reactions are carried out by complex molecular machines,
containing multiple subunits, that carry out the diverse biochemical activities required for
each process.
Replication and transcription differ in a number of ways, however. First, of course, is
that transcription produces an RNA copy of the sequence, whereas replication produces a
DNA copy. Also, replication generates a single copy of the entire genome, whereas
transcription produces multiple copies of specific, limited sections of the genome. Another
difference is that replication initiates at multiple locations that, in some cases at least (in
particular multi-cellular eukaryotes) have flexible sequence requirements, whereas
transcription begins (and stops) at very precise sequences. Also, at a mechanistic level, DNA
replication requires a primer sequence, and the new DNA strand remains hybridized to the
template, whereas transcription can begin de novo and the new RNA is displaced from the
template. Finally, replication contains multiple proofreading mechanisms, giving rise to a
very high level of accuracy, whereas transcription has fewer, less stringent methods of
proofreading and is correspondingly less accurate.
2.
DNA replication is more accurate than transcription because of its multiple, rigorous
The three basic phases of transcription are initiation, elongation, and termination. In
initiation, RNA polymerase binds to the promoter (along with other factors), leading to the
unwinding of the DNA at the start site and initial synthesis (in a 5' to 3' direction) of an RNA,
using one of the DNA strands as a template. The elongation phase of transcription begins
when the polymerase synthesizes a short stretch of RNA and clears the promoter. During
elongation, the polymerase moves along the gene, synthesizing RNA as it goes, and also
unwinds the DNA ahead of the polymerase, separates the growing RNA chain from the DNA,
proofreads the RNA, and re-anneals the DNA behind the enzyme. During the termination
phase, the polymerase stops synthesizing RNA, releases the transcript, and leaves the DNA
template.
Initiation, the most common locus of regulatory action, can be targeted in any of a
number of ways. For example, many regulators affect the binding of polymerase to the
promoter, and numerous others modulate the ability of the polymerase to unwind the DNA
and initiate RNA synthesis. Elongation is a less common target for regulation, although a
number of factors do affect this stage of transcription. For example, certain regulators act on
the ability of polymerase to clear the promoter once a short transcript has been made. Other
elements modulate various properties of the polymerase during elongation, such as its
processivity (i.e. the likelihood that it will fall off the template), its rate of movement along
the DNA template, or its proofreading activity. Finally, regulators that act on termination can
target the processing of the transcript (such as polyadenylation), the cleavage of the RNA,
and the termination of RNA synthesis by the polymerase enzyme.
4.
The core bacterial polymerase contains two alpha subunits, one beta, one beta', and
one omega subunit. The holoenzyme includes the core polymerase as well as a sigma factor.
The overall shape of the enzyme resembles a crab claw, with the pincers made up mostly of
the two largest subunits, beta and beta'.
The sigma factor, which extends away from the holoenzyme, mediates the binding of
RNA polymerase to the promoter. For example, 70, the most common E. coli sigma factor,
binds to the 10 and 35 elements of promoters the promoter, specifically through its C
terminal domain (which also extends away from the through its region 2 and region 4,
respectively. The alpha subunit also makes contacts with enzyme), which recognizes the UP
promoter element.
5.
The transition to an open complex involves structural changes in both the RNA
polymerase and the DNA at the promoter. In bacteria, for example, the "pincers" at the front
of the enzyme clamp down on the DNA, and the sigma subunit shifts so that it no longer
blocks DNA accessing the active site of the enzyme. The promoter, at the same time,
unwinds, allowing access to the single stranded template and nontemplate strands.
The transition to an open complex is critical for replication initiation because, first, it
allows access to the template strand, but also because it represents an irreversible step
towards initiation. The energetics of the transition are such that a closed complex will never
revert to the open form, but will instead proceed irreversibly to initiation.
The transition to the open complex, and concomitant unwinding of the DNA, requires
no outside source of energy in bacteria. Instead, the reaction is driven by a spontaneous
conformational change in the DNA-polymerase complex. Unwinding of the DNA in
eukaryotes, in contrast, requires hydrolysis of ATP, specifically by the helicase-like TFIIH
factor.
7.
RNA polymerase has clear affinity for promoters, as it binds specifically to them to
initiate transcription. Because of that affinity, it would tend to stay put unless other equally
strong forces pull the polymerase away from the promoter, or if the interaction between the
polymerase and the promoter were altered in a way that decreased the affinity. As it happens,
both of these appear to play a role in promoter clearance. First, the polymerase is pulled away
from the promoter by RNA synthesis itself, which involves a significant loss of free energy.
But the interaction between the polymerase and the promoter is also altered during the shift to
elongation. In bacteria, for example, the sigma subunitwhich makes the most extensive
contacts with the promoteris shed upon passage into the elongation phase. Without the
sigma-mediated interactions with the promoter, the polymerase has much less affinity for the
promoter. Similarly, in eukaryotes, the phosphorylation of the CTD decreases the affinity of
the polymerase for the promoter, because the phosphorylation diminishes the interaction
between the polymerase and promoter-bound initiation factors.
8.
The polymerase keeps all of the strands (and individual nucleotides) separate by
threading each of them through a distinct part of the enzyme. The bacterial holoenzyme, for
example, has five different channels for keeping each of the involved polynucleotides and
nucleotides separate. Ribonucleotides enter the enzyme through a particular NTP-uptake
channel; an RNA-exit channel permits the growing RNA chain to leave the enzyme; the
downstream DNA channel allows double-stranded DNA ahead of the enzyme to enter, and
two different channels passing through the enzyme are dedicated to the single-stranded
template and nontemplate strands.
9.
including the transition from initiation to elongation, as well as termination. When Pol II
initially binds to the promoter, CTD is unphosphorylated, and interacts with initiation factors
bound to the promoter. The tail is subsequently phosphorylated at multiple sites by a kinase
present in TFIIH (and other kinases). This phosphorylation diminishes the interaction
between the polymerase and the initiation factors, and instead promotes new interactions with
other factors involved in elongation and RNA processing. Accordingly, the phosphorylation
of the CTD takes place in concert with the shift to elongation, and is associated with a
replacement of the polymerase-bound initiation factors with elongation/termination factors.
Pol I and Pol III can likely function without the CTD because of the major differences
in the way they are regulated in comparison to Pol II. For example, the CTD contributes to
the interactions between Pol II and initiation factors, but Pol I and Pol III rely on different
initiation factors than Pol II. Also, CTD is involved in interactions with the Mediator
Complex at Pol II promoters, whereas neither pol I nor pol III rely on the Mediator for
transcription initiation.
11.
DNA present in vivo differs from that used in in vitro systems because it is packaged
into nucleosomes and higher order chromatin structures. These can interfere with the stable
binding of polymerase and other transcription factors to the DNA, necessitating the presence
of additional factors that can help promote polymerase binding. Mediator does just that,
helping DNA-bound transcriptional activators interact with RNA polymerase to stabilize its
binding to the DNA.
12.
The elongation factors can help promote elongation in several ways. For example,
some can help get elongation started in the first place, such as the CTD-phosphorylating
activity of the P-TEFb kinase. Others can help speed up elongation, such as TFIIS and its role
in limiting the pausing of the polymerase at certain sequences. Some factors can promote
elongation by enhancing the processivity of the RNA polymerasethat is, the stability of the
interaction between the polymerase and the DNA template. Finally, certain factors can help
stimulate the proofreading activity of polymerase.
The elongation factors are recruited to the polymerase through CTD. Specifically,
whereas general transcription factors have affinity for the unphosphorylated CTD, elongation
factors have affinity for the phosphorylated form of the tail. Accordingly, phosphorylation of
the CTD leads to the shedding of the general transcription factors and their replacement with
elongation factors.
13.
mRNA is processed in three major ways prior to being exported from the nucleus: 5'
capping of the transcript, splicing, and 3' polyadenylation. In 5' capping, a modified guanine
base is added to the 5' end of the transcript. Splicing involves the removal of introns from the
transcript to generate the mature mRNA. Finally, 3' polyadenylation of the message involves
cleavage of the RNA and the addition of numerous adenine residues at the 3' end.
These processing events are intricately linked to the polymerase's progress through
elongation and termination. For example, the elongation factor hSPT5 recruits the capping
enzyme to the mRNA early during transcription, and the capping machinery later dissociates
when Ser5 of the CTD becomes dephosphorylated. Another elongation factor, TAT-SF1, helps
recruit certain components of the splicing machinery. Recruitment of the splicing machinery
is also triggered by phosphorylation of Ser2 within the CTD. Finally, 3' polyadenylation and
RNA cleavage is triggered by CPSF and CstF, which travel along with the CTD during
elongation, only to leave the polymerase and move onto the RNA when the polymerase
reaches certain specific sequences at the 3' end of the gene.