|Content of Nucleic Acid Synthesis
Transcription is a highly complex process because of its
defined initiation and termination sites in the genome and
the subsequent processing and regulation of its synthesis.
The steady-state level of a protein in the cell is the
balance of its rate of synthesis and degradation. The synthesis
is determined primarily by the steady-state level of
its mRNA. Thus, the rate of transcription often determines
the level of its gene product in vivo.
As mentioned earlier,RNAsynthesis is catalyzed by the
RNA polymerase in all organisms. Prokaryotes express a
single RNA polymerase used for synthesis of all RNAs,
while eukaryotes encode multiple RNA polymerases with
dedicated functions. RNA polymerase I (Pol I) in eukaryotic
cells is responsible for synthesis of ribosomal RNA,
which accounts for more than 70% of total RNA in the
cell. Pol III catalyzes synthesis of small RNA molecules,
including transferRNAs which bring in appropriate amino
acids to the ribosome for protein synthesis by using their
“anti-codon” triplet bases. Pol II is responsible for synthesis
of all other RNA, specifically mRNA.
RNA polymerases of all organisms are complex machines
consisting of multiple subunits which alter conformation.
A variety of structural analyses show the presence
of a 2.5-nm-wide “channel” on the surface of all
DNA polymerases which could be the path for DNA.
The RNA polymerase holoenzyme binds to a promoterspecific
recognition sequence upstream (5´ side of the transcribed
strand) of the site of synthesis initiation. While the
RNA polymerase is normally present as a closed complex
with nonspecific DNA, in which DNA base pairs are not
broken, a significant conformational change produces the
open complex whenRNAthe enzyme binds the promoter,
unwinds the DNA duplex, and is poised to initiate RNA
As in the replication process, initiation is the first stage
in transcription and denotes the formation of first phosphodiester
bond. Unlike in the case of DNA synthesis, RNA
chains are initiated de novo without the need of a primer.
However, when a primer oligonucleotide is present, RNA
polymerases can also extend the primer as dictated
by the template strand. A purine nucleotide invariably
starts the RNA chains in both prokaryotes and eukaryotes,
and the overall rate of chain growth is about
40 nucleotides per second at 37◦C in E. coli. This rate
is much slower than that for DNA chain elongation
(∼800 base pairs per second at 37◦ for the E. coli genome).
RNA synthesis is not monotonic, andRNApolymerases
can move backward like DNA polymerases do for their
editing function in which an incorrectly inserted deoxynucleotide
is removed by 3´ exonuclease activity. RNA polymerases
stall, back track, and then cleave off multiple
newly inserted nucleotides at the 3´ terminus. Subsequently,
polymerases move forward along the DNA template
and resynthesize the cleaved region. Based on the
segment of DNA covered by an RNA polymerase as analyzed
by DNA footprinting, it has been proposed that the
enzyme alternatively compresses and extends in its binding
to the DNA template and acts like an inchworm in its
RNA polymerases of both prokaryotes and eukaryotes
function as complexes consisting of a number of subunits.
The E. coli RNA polymerase enzyme with a total molecular
mass of about 465 kD contains two α-subunits, one β-
and one β´-subunit each, and a σ-subunit which provides
promoter specificity. During chain elongation, a ternary
complex of macromolecules among DNA template, RNA
polymerase, and nascent RNA is maintained in which
most of the nascent RNA molecule is present in a singlestranded
unpaired form. The stability of the complex is
maintained by about nine base pairs between RNAand the
transcribed (noncoding) DNA strand at the growing point.
While DNA replication warrants permanent unwinding
of the parental duplex DNA, asymmetric copying of only
one strand by RNA polymerase requires localized strand
separation which is induced by the polymerase itself, resulting
in a transcription bubble. During chain elongation,
this bubble moves along the DNA duplex. Initiation
of RNA synthesis is enhanced in an in vitro reaction with
supercoiled duplex circular DNA template in which base
pairs are destabilized due to torsional stress. Unwinding
of the helix at the transcription site causes overwinding
(positive supercoiling) of the template DNA ahead of the
transcription bubble and underwinding (negative supercoiling)
behind the bubble.