4:  4.3 Nucleic Acid Synthesis

Both DNA and RNA Chains Are Produced by Copying of Template DNA Strands

The regular pairing of bases in the double-helical DNA structure suggested to Watson and Crick a mechanism of DNA synthesis. Their proposal that new strands of DNA are synthesized by copying of parental strands of DNA has proved to be correct.

The DNA strand that is copied to form a new strand is called a template. The information in the template is preserved: although the first copy has a complementary sequence, not an identical one, a copy of the copy produces the original (template) sequence again. In the replication of a double-stranded, or duplex, DNA molecule, both original (parental) DNA strands are copied. When copying is finished, the two new duplexes, each consisting of one of the two original strands plus its copy, separate from each other. In some viruses, single-stranded RNA molecules function as templates for synthesis of complementary RNA or DNA chains (Chapter 7). However, the vast majority of RNA and DNA in cells is synthesized from preexisting duplex DNA.

Nucleic Acid Strands Grow in the 5′ → 3′ Direction

All RNA and DNA synthesis, both cellular and viral, proceeds in the same chemical direction: from the 5′ (phosphate) end to the 3′ (hydroxyl) end (see Figure 4-13). Nucleic acid chains are assembled from 5′ triphosphates of ribonucleosides or deoxyribonucleosides. Strand growth is energetically unfavorable but is driven by the energy available in the triphosphates. The α phosphate of the incoming nucleotide attaches to the 3′ hydroxyl of the ribose (or deoxyribose) of the preceding residue to form a phosphodiester bond, releasing a pyrophosphate (PP_i). The equilibrium of the reaction is driven further toward chain elongation by pyrophosphatase, which catalyzes the cleavage of PP_i into two molecules of inorganic phosphate (see Table 2-7).

RNA Polymerases Can Initiate Strand Growth but DNA Polymerases Cannot

The enzymes that copy (replicate) DNA to make more DNA are DNA polymerases; those that copy (transcribe) DNA to form RNA are RNA polymerases. Because the two DNA strands are complementary, rather than identical, transcription of a particular DNA segment theoretically could yield two mRNAs with different sequences and hence different protein-coding potentials. Generally, only one strand of the duplex in a particular DNA segment gives rise to usable information when transcribed into mRNA. In unusual cases, though, limited sections of DNA encode proteins on both strands.

An RNA polymerase can find an appropriate initiation site on duplex DNA; bind the DNA; temporarily “melt,” or separate, the two strands in that region; and begin generating a new RNA strand (Figure 4-15). As discussed in Chapter 10, the location and regulated use of transcription start sites to produce mRNA requires many dozens of proteins in eukaryotes and several proteins even in bacteria. The nucleotide at the 5 ′ terminus of a growing RNA strand is chemically distinct from the nucleotides within the strand in that it retains all three phosphate groups. When an additional nucleotide is added to the 3′ end of the growing strand, only the α-phosphate is retained; the β and γ phosphates are lost as pyrophosphate, which is subsequently hydrolyzed to yield 2 molecules of inorganic phosphate.

Unlike RNA polymerases, DNA polymerases cannot initiate chain synthesis de novo; instead, they require a short, preexisting RNA or DNA strand, called a primer, to begin chain growth. With a primer base-paired to the template strand, a DNA polymerase adds nucleotides to the free hydroxyl group at the 3′ end of the primer:

If RNA is the primer, the polynucleotide copied from the template is RNA at the 5′ end and DNA at the 3′ end.

Both prokaryotic and eukaryotic cells have several different types of DNA polymerases. Some polymerases participate in making new DNA to prepare for cell division; other polymerases serve in the repair and recombination of DNA molecules. The structure, mechanism, and physiological role of these enzymes are described in Chapter 12.

Replication of Duplex DNA Requires Assembly of Many Proteins at a Growing Fork

Because duplex DNA consists of two intertwined strands, the base-pair copying of each strand requires unwinding of the original duplex, which is accomplished by specific “unwinding proteins” called helicases. As noted earlier, local unwinding of duplex DNA produces torsional stress, leading to formation of supercoils, which are removed by topoisomerases. The action of all these proteins produces a moving, highly specialized region of the DNA called the growing fork, at which DNA polymerase carries out nucleotide addition. In order for DNA polymerase to move along and copy a duplex DNA, helicase must sequentially unwind the duplex and topoisomerase must remove the supercoils that form.

DNA replication begins with creation of a growing fork by a protein or proteins that have helicase activity and unwind a short section of parental DNA. A specialized RNA polymerase then forms short RNA primers complementary to the unwound template strands. Each such primer, still bound to its complementary DNA strand, is then elongated by DNA polymerase, thereby forming a new daughter strand. One final major complication in the operation of a DNA growing fork is that although the two strands of the parental duplex are antiparallel, nucleo-tides can be added to the growing new strands only in the 5′ → 3′ direction. As diagrammed in Figure 4-16, synthesis of one daughter strand, called the leading strand, proceeds continuously from a single RNA primer in the 5′ → 3′ direction, the same direction as movement of the growing fork. Because growth of the other daughter strand, called the lagging strand, also must occur in the 5′ → 3′ direction, copying of its template strand must somehow occur in the opposite direction from the movement of the growing fork. A cell accomplishes this feat by producing additional short RNA primers every 1000 bases or so on the second parental strand, as more of the strand is exposed by unwinding. Each of these primers, base-paired to their template strand, is elongated in the 5′ → 3′ direction, forming discontinuous segments called Okazaki fragments after their discoverer Reiji Okazaki. The RNA primer of each Okazaki fragment is removed and replaced by DNA chain growth from the neighboring Okazaki fragment; finally an enzyme called DNA ligase joins the adjacent fragments. At least 30 proteins participate in the formation and operation of a DNA growing fork; this DNA-replication machine is discussed in detail in Chapter 12.

Organization of Genes in DNA Differs in Prokaryotes and Eukaryotes

Having outlined the principles governing the stepwise assembly of polynucleotides, we now focus briefly on the large-scale arrangement of information in DNA and how this arrangement dictates the requirements for RNA manufacture so that information transfer goes smoothly. The simplest definition of a gene is a “unit of DNA that contains the information to specify synthesis of a single polypeptide chain.” The number of genes in cells varies widely, with the simpler non-nucleated prokaryotic cells having far fewer genes than eukaryotic cells. The vast majority of genes carry information to build protein molecules, and it is the RNA copies of such protein-coding genes that are the mRNA molecules of cells. In recent years, the entire sequence of the DNA genome of several organisms has been determined, providing direct evidence for large differences in their protein-coding capacity (Chapter 7).

The most common arrangement of protein-coding genes in all prokaryotes has a powerful and appealing logic: genes devoted to a single metabolic goal, say, the synthesis of the amino acid tryptophan, are most often found in a contiguous array in the DNA. This gene order makes it possible to produce a continuous strand of mRNA that carries the message for a related series of enzymes devoted to making tryptophan (Figure 4-17a). Each section of the mRNA represents the unit (or gene) that instructs the protein-synthesizing apparatus to make a particular protein. Such an arrangement of genes in a functional group is called an operon, because it operates as a unit from a single transcription start site. In prokaryotic DNA the genes are closely packed with very few noncoding gaps, and the DNA is transcribed directly into colinear mRNA, which then is translated into protein, even while stretches of the mRNA closer to the 3′ end are still being produced.

This economic clustering of genes devoted to a single metabolic function does not occur in eukaryotes, even simple ones like yeasts that can be metabolically similar to bacteria. Rather, eukaryotic genes, even those devoted to a single pathway, are most often physically separated in the DNA, sometimes even being located on different chromosomes. Each gene is transcribed from its own start site, producing one mRNA, which generally is translated to yield a single protein (Figure 4-17b). Moreover, when researchers first compared the nucleotide sequences of eukaryotic mRNAs with the DNAs encoding them, they were astounded to find that the uninterrupted protein-coding sequence of a given mRNA was broken up (discontinuous) in its corresponding section of DNA. They concluded that the eukaryotic gene existed in pieces of coding sequence, the exons, separated by non-protein-coding segments, the introns. This astonishing finding, first discovered in viruses that infect eukaryotic cells, implied that the long initial RNA copy, called the primary transcript, the entire copied DNA sequence, had to be clipped apart to remove the introns and then carefully stitched back together to produce many mRNAs of eukaryotic cells.

Eukaryotic Primary RNA Transcripts Are Processed to Form Functional mRNAs

In prokaryotic cells, which have no nuclei, translation of an mRNA into protein can begin from the 5′ end of the mRNA even while the 3′ end is still being copied from DNA. Thus, transcription and translation can occur concurrently. In eukaryotic cells, however, not only is the nucleus separated from the cytoplasm where protein synthesis occurs, but the primary RNA transcript of a protein-coding gene must undergo several modifications, collectively termed RNA processing, that yield a functional mRNA. This mRNA then must be transported to the cytoplasm before it can be translated into protein. Thus, transcription and translation cannot occur concurrently in eukaryotic cells.

The initial steps in processing of all eukaryotic primary RNA transcripts occur at the two ends, and these modifications are retained in mRNAs. To the initiating (5′) nucleotide of the primary transcript is added the 5′ cap, which may serve to protect mRNA from enzymatic degradation (Figure 4-18). This modification occurs before transcription is complete, so the 5′ cap is present in the primary transcript. Processing at the 3′ end of the primary transcript involves cleavage by an endonuclease to yield a free 3′-hydroxyl group to which a string of adenylic acid residues is added by an enzyme called poly(A) polymerase. The resulting poly(A) tail contains 100 – 250 bases, being shorter in yeasts and invertebrates than in vertebrates. Poly(A) polymerase is part of a complex of proteins that adds the poly(A) tail. This complex does not require a template and can determine the correct number of A residues to add in each species.

The final step in the processing of many different eukaryotic mRNA molecules is splicing: the internal cleavage of the RNA transcript to excise the introns, followed by ligation of the coding exons. Many eukaryotic mRNAs also contain noncoding regions at each end; these are referred to as the 5′ and 3′ untranslated regions (UTRs). Figure 4-19 summarizes the basic steps in RNA processing. We examine the cellular machinery for carrying out processing of mRNA, as well as tRNA and rRNA, in Chapter 11.

SUMMARY

• The transfer of information from genes to proteins is assisted by proteins that participate in the synthesis of DNA and RNA.

• Polynucleotide and polypeptide chains are assembled from a limited number of monomeric units that are added one at a time, beginning at the 5′ end in nucleic acids and the amino-terminal end in proteins (see Figure 4-13). In both cases, the initial polymeric product generally is modified in some fashion to produce a functional molecule.

• A polynucleotide chain is synthesized by copying of a complementary template strand (usually DNA). In this process, the duplex DNA is locally unwound, revealing the unpaired template strand, and nucleotides are added to the 3′-hydroxyl end of the growing strand by RNA or DNA polymerase.

• RNA polymerase can initiate transcription of DNA into RNA by binding to a specific start site and unwinding the duplex. As the enzyme moves along the DNA, it unwinds sequential segments of the DNA and adds nucleotides to the growing RNA strand (see Figure 4-15). Most commonly, only one DNA strand in any one locus is transcribed into RNA.

• Replication of DNA requires the assistance of helicase to unwind the duplex, topoisomerase to remove supercoils, and a specialized RNA polymerase to form RNA primers because DNA polymerase cannot start chains. Nucleotide addition at the growing fork, a moving region of strand separation produced by sequential unwinding of the duplex, is catalyzed by one type of DNA polymerase.

• During DNA replication, the two new daughter strands are assembled somewhat differently because DNA polymerase can add nucleotides only in the 5′ → 3′ direction (see Figure 4-16). One new strand, the leading strand, is elongated continuously from a single primer. The other new strand, the lagging strand, is synthesized discontinuously as a series of short segments, called Okazaki fragments, initiated from multiple RNA primers. After removal of the intervening primer, adjacent Okazaki fragments are joined by DNA ligase.

• In prokaryotic DNA, related protein-coding genes are clustered into a functional region, an operon, which is transcribed from a single start site into one mRNA encoding multiple proteins (see Figure 4-17a). Translation of a mRNA can begin before synthesis of the mRNA is complete.

• In eukaryotic DNA, each protein-coding gene is transcribed from its own start site, and very often the coding regions (exons) are separated by noncoding regions (introns). The primary RNA transcript produced from such a gene must undergo processing to yield a functional mRNA. During processing, the ends of all primary transcripts are modified by addition of a 5′ cap and 3′ poly(A) tail; many transcripts also undergo splicing — removal of the introns and joining of the exons (see Figure 4-19).