Similarity of DNA and RNA Synthesis

All nucleic acids are usually synthesized by DNA template- guided polymerization of nucleotides—ribonucleotides for RNA and deoxy(ribo)nucleotides for DNA. The reactant monomers are 5´ ribonucleoside (or deoxyribonucleoside) triphosphates. These can be described in the following chemical equations:

DNA + ndNTP DNA + nPPi

and

(DNA) + nNTP (DNA) + RNA + nPPi

Enzymatic polymerization is carried out by DNA and RNA polymerases, both of which carry out pyrophosphorolysis, i.e., cleavage of a high energy pyrophosphate bond coupled to esterification of 5´ phosphate linked to the 3´-OH of the previous residue. The reaction is reversible, although it strongly favors synthesis. Degradation of nucleic acids is not due to reversal of the reaction, but rather a hydrolytic reaction catalyzed by nucleases, namely, RNases and DNases, which generate nucleotides or deoxynucleotides, respectively.

Three distinct stages are involved in the biosynthesis of both DNA and RNA: initiation, chain elongation, and termination. Initiation denotes de novo synthesis of a nucleic acid polymer which is generally well regulated by complex processes, as described later. The key difference in initiation of a DNA vs RNA chain is that RNA polymerases can start a new chain, while all DNA polymerases require a “primer,” usually a short RNA or DNA sequence with a 3´-OH terminus, to which the first deoxynucleotide residue is added. Elongation denotes continuing polymerization of the monomeric nucleotides, and termination defines stoppage of nucleotide addition to the growing polymer chain.

During synthesis the enzymes catalyzing the polymerization reaction are guided by nucleic acid templates that provide the complementary sequence for the incorporated nucleotides (Fig. 4). The basic catalytic enzyme in such reactions is called DNA or RNA polymerase. In cells the template for both DNA and RNA is genomic DNA. There are some exceptions to these general rules. Some DNA polymerases can synthesize homo- or heteropolymers of deoxynucleotides in vitro in the absence of a template; the substrate is restricted to one or two dNTPs. While it is unlikely that these homo- or heteropolymers, e.g., (dAdT)_n or poly(dA)_npoly(dT)_n, are formed in vivo, the availability of these polymers significantly advanced our understanding of the properties of DNA, before the age of chemical or enzymatic oligonucleotide synthesis.

There are some exceptions to the norm of DNAdependent DNA or RNA synthesis, mostly in lower eukaryotes or viruses (Fig. 5). One example is RNAdependent RNA synthesis in plant, animal, or bacterial viruses. In these cases, a single-stranded RNA template rather than double-stranded DNA guides synthesis of the complementary RNA strand, based on conventional base pairing. The polarity of RNA adds a level of complexity during synthesis. Thus, the RNA genome of a virus that can be directly read and thus provides the mRNA function is called the positive strand, as in polio virus. In this case, the viral genome RNA functions as the mRNA and encodes the RNA polymerase, which is synthesized like other viral proteins in the infected cell. This RNA polymerase subsequently synthesizes the complementary negative strand, which then serves as the template for synthesis of the progeny positive strand RNA. The progeny RNA is then packaged into mature progeny virus.

In contrast, the genomic RNA of negative strand viruses (e.g., vesicular stomatitis virus) cannot function directly as mRNA and thus cannot guide synthesis of proteins, including the RNA replicase, by itself after the infection of host cells. These viruses carry their own RNA replicase within the virion capsids, which carry out (+) mRNA strand synthesis after infection (Fig. 5).

Retroviruses comprise diverse groups of viruses, including human immunodeficiency virus (HIV), which share a common mechanism of genome replication. The RNA genomes of these viruses encode an RNA dependent DNA polymerase (reverse transcriptase or RT) which first generates the complementary (c) DNA of the viral genome. RT has also RNaseH (specific nuclease for degrading RNA from RNA–DNA hybrids) and DNAdependent DNA polymerase activities. After copying the RNA template, the enzyme degrades the RNA and is able to convert the resulting single-stranded cDNA to duplex DNA. This is then integrated into the host cell genome as proviral DNA, from which the progeny viral RNA is eventually transcribed. Thus, the reverse transcriptase is an unusual polymerase because it can utilize both RNA and DNA templates (Fig. 5). There is strong evidence that such reverse transcription was involved in synthesis of “retrotransposons,” a special class of mobile genetic elements, during the evolution of mammalian genomes. These mobile genetic elements, also known as transposons, when identified in bacteria and lower eukaryotes, consist of specific DNA sequences which can be relocated randomly in the genome. The transposition is mediated by enzymes called transposase, usually synthesized by a gene in the transposon. During transposition of retransposons, certain mRNAs are reverse transcribed and then integrated into the genome like the proviral sequence. The presence of specific flanking sequences allows these elements to relocate to other sites in the genome.

FIGURE 4 Replication of circular DNA of prokaryotes and viruses, plasmids, and mitochondria. The basic steps of replication are shown. (A) Rolling circle mode of replication for singlestranded circular DNA: single-stranded (ss) DNA is replicated to the replicative form (RF), which then acts as the template for progeny ss DNA synthesis via a rolling circle intermediate. (B) Circular duplex DNA can be replicated at the ori site by formation of a θ intermediate. Replication could be bidirectional (as shown here) or unidirectional. 5´→3´ chain growth dictates that DNA synthesis is continuous on one side of the ori and discontinuous on the other side for each strand; (+) and (−) strands are shown to distinguish the strand types. (C) Replication of a linear genome with multiple origins.

) which is present in the virus particle. Once the complementary (+) strand which serves as mRNA is synthesized, viral-specific proteins are synthesized, including RNA replicase. (C) Replication of (+) stranded retroviral genomes first involves synthesis of the reverse transcriptase which directs synthesis of duplex DNA in two stages from the RNA template. Circularization of the DNA followed by its genomic integration allows synthesis of progeny viral RNA by the host transcription machinery." width="352" height="366" />

FIGURE 5 Replication of mammalian viral RNA genome. The basic steps of replication are shown for (A) a (+) strand genome, which acts as an mRNA for encoding viral proteins; (B) a (−) viral genome cannot encode protein and first has to be replicated by the RNA replicase () which is present in the virus particle. Once the complementary (+) strand which serves as mRNA is synthesized, viral-specific proteins are synthesized, including RNA replicase. (C) Replication of (+) stranded retroviral genomes first involves synthesis of the reverse transcriptase which directs synthesis of duplex DNA in two stages from the RNA template. Circularization of the DNA followed by its genomic integration allows synthesis of progeny viral RNA by the host transcription machinery.