Page 131 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

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Nucleic Acid Synthesis 869

first requirement is the sequence information for the ter- RNA polymerases of all organisms are complex ma-
mini of the segment, based on which the oligonucleotides chines consisting of multiple subunits which alter confor-
will be designed for each terminus and then synthesized. mation. A variety of structural analyses show the pres-
However, errors of replication cannot be completely elim- ence of a 2.5-nm-wide “channel” on the surface of all
inated. Any error in DNA synthesis that occurs early DNA polymerases which could be the path for DNA.
will be perpetuated. Furthermore, if replication is initi- The RNA polymerase holoenzyme binds to a promoter-

ated by primers annealed to an incorrect DNA sequence, specific recognition sequence upstream (5 side of the tran-
the wrong PCR product will be generated. scribed strand) of the site of synthesis initiation. While the
Primarily, because it has both sensitivity and speci- RNA polymerase is normally present as a closed complex
ficity, PCR technology has revolutionized many aspects with nonspecific DNA, in which DNA base pairs are not
of biomedical research. Several modifications of the basic broken, a significant conformational change produces the
methodology have provided additional powerful tools. open complex when RNA the enzyme binds the promoter,
For example, a trace amount of RNA can be quantitated unwinds the DNA duplex, and is poised to initiate RNA
by reverse transcriptase PCR (RTPCR), where a reverse synthesis.
transcriptase synthesizes the complementary DNA strand As in the replication process, initiation is the first stage
of the RNA, which then serves as the template for regular intranscriptionanddenotestheformationoffirstphospho-
PCR. diester bond. Unlike in the case of DNA synthesis, RNA
DNA in a very small amount of biological samples can chains are initiated de novo without the need of a primer.
be amplified by PCR. This technique has been exploited However, when a primer oligonucleotide is present, RNA
in criminal investigations to identify suspects by “finger- polymerases can also extend the primer as dictated
printing” their DNA, which involves determining a char- by the template strand. A purine nucleotide invariably
acteristic pattern of repeat sequences in the genome after starts the RNA chains in both prokaryotes and eukary-
PCR amplification of the total DNA. PCR has also been otes, and the overall rate of chain growth is about
utilized in the identification of pathogens and other mi- 40 nucleotides per second at 37 Cin E. coli. This rate
◦
croorganisms, based on certain unique sequences of each is much slower than that for DNA chain elongation
organism. PCR has been exploited for a variety of in vitro (∼800 base pairs per second at 37 for the E. coli genome).
◦
manipulations of DNA sequences in plasmids, viruses, RNA synthesis is not monotonic, and RNA polymerases
and synthetic DNA by generating site-specific mutations can move backward like DNA polymerases do for their
and a variety of recombinant DNA plasmids. editing function in which an incorrectly inserted deoxynu-
cleotide is removed by 3 exonuclease activity. RNA poly-

merases stall, back track, and then cleave off multiple
VI. TRANSCRIPTIONAL PROCESSES newly inserted nucleotides at the 3 terminus. Subse-

quently, polymerases move forward along the DNA tem-
Transcription is a highly complex process because of its plate and resynthesize the cleaved region. Based on the
defined initiation and termination sites in the genome and segment of DNA covered by an RNA polymerase as ana-
the subsequent processing and regulation of its synthe- lyzed by DNA footprinting, it has been proposed that the
sis. The steady-state level of a protein in the cell is the enzyme alternatively compresses and extends in its bind-
balance of its rate of synthesis and degradation. The syn- ing to the DNA template and acts like an inchworm in its
thesis is determined primarily by the steady-state level of transit.
its mRNA. Thus, the rate of transcription often determines RNA polymerases of both prokaryotes and eukaryotes
the level of its gene product in vivo. function as complexes consisting of a number of subunits.
As mentioned earlier, RNA synthesis is catalyzed by the The E. coli RNA polymerase enzyme with a total molecu-
RNA polymerase in all organisms. Prokaryotes express a lar mass of about 465 kD contains two α-subunits, one β-

single RNA polymerase used for synthesis of all RNAs, and one β -subunit each, and a σ-subunit which provides
while eukaryotes encode multiple RNA polymerases with promoter specificity. During chain elongation, a ternary
dedicated functions. RNA polymerase I (Pol I) in eukary- complex of macromolecules among DNA template, RNA
otic cells is responsible for synthesis of ribosomal RNA, polymerase, and nascent RNA is maintained in which
which accounts for more than 70% of total RNA in the most of the nascent RNA molecule is present in a single-
cell. Pol III catalyzes synthesis of small RNA molecules, stranded unpaired form. The stability of the complex is
including transfer RNAs which bring in appropriate amino maintained by about nine base pairs between RNA and the
acids to the ribosome for protein synthesis by using their transcribed (noncoding) DNA strand at the growing point.
“anti-codon” triplet bases. Pol II is responsible for syn- While DNA replication warrants permanent unwinding
thesis of all other RNA, specifically mRNA. of the parental duplex DNA, asymmetric copying of only