Nucleotide Structure and Sequence

DNA consists of deoxyribose sugars connected by phosphodiester bonds (Figure 2-2, A). The bases that are covalently linked to each deoxyribose sugar are the key to the genetic code within the DNA molecule. The four bases include two purines, adenine (A) and guanine (G), and the two pyrimidines, cytosine (C) and thymine (T) (Figure 2-3). In RNA, uracil replaces thymine. The combined sugar, phosphate, and a base form a single unit referred to as a nucleotide (adenosine triphosphate [ATP], guanine triphosphate [GTP], cytosine triphosphate [CTP], and thymine triphosphate [TTP]). DNA and RNA are nucleotide polymers (i.e., chains or strands), and the order of bases along a DNA or RNA strand is known as the base sequence. This sequence provides the information that codes for the proteins that will be synthesized by microbial cells; that is, the sequence is the genetic code.

DNA Molecular Structure

The intact DNA molecule is composed of two nucleotide polymers. Each strand has a 5’ (prime) phosphate and a 3’ (prime) hydroxyl terminus (see Figure 2-2, A). The two strands run antiparallel, with the 5’ of one strand opposed to the 3’ terminal of the other. The strands are also complementary, because the adenine base of one strand always binds to the thymine base of the other strand by means of two hydrogen bonds. Similarly, the guanine base of one strand always binds to the cytosine base of the other strand by means of three hydrogen bonds. As a result of the molecular restrictions of these base pairings, along with the conformation of the sugar-phosphate backbones oriented in antiparallel fashion, DNA has the unique structural conformation often referred to as a “twisted ladder” or double helix (see Figure 2-2, B). Additionally, the dedicated base pairs provide the format essential for consistent replication and expression of the genetic code. In contrast to DNA, which carries the genetic code, RNA rarely exists as a double-stranded molecule. The three major types of RNA (messenger RNA [mRNA], transfer RNA [tRNA], and ribosomal RNA [rRNA]) play key roles in gene expression.

Genes and the Genetic Code

A DNA sequence that encodes for a specific product (RNA or protein) is defined as a gene. Thousands of genes in an organism encode messages or blueprints for the production of one or more proteins and RNA products that play essential metabolic roles in the cell. All the genes in an organism comprise the organism’s genome. The size of a gene and an entire genome is usually expressed in the number of base pairs (bp) present (e.g., kilobases [10³ bases], megabases [10⁶ bases]).

Certain genes are widely distributed among various organisms while others are limited to particular species. Also, the base pair sequence for individual genes may be highly conserved (i.e., show limited sequence differences among different organisms) or be widely variable. As discussed in Chapter 8, these similarities and differences in gene content and sequences are the basis for the development of molecular tests used to detect, identify, and characterize clinically relevant microorganisms.

Chromosomes

The genome is organized into discrete elements known as chromosomes. The set of genes within a given chromosome is arranged in a linear fashion, but the number of genes per chromosome is variable. Similarly, although the number of chromosomes per cell is consistent for a given species, this number varies considerably among species. For example, human cells contain 23 pairs (i.e., diploid) of chromosomes whereas bacteria contain a single, unpaired (i.e., haploid) chromosome.

Bacteria are classified as prokaryotes; therefore, the chromosome is not located in a membrane-bound organelle (i.e., nucleus). The bacterial chromosome contains the genes essential for viability and exists as a double-stranded, closed, circular macromolecule. The molecule is extensively folded and twisted (i.e., supercoiled) in order to fit within the confined space of the bacterial cell. The linearized, unsupercoiled chromosome of the bacterium Escherichia coli is about 1300 µm long, but it fits within a cell 1 × 3 µm; this attests to the extreme compact structure of the supercoiled bacterial chromosome. For genes in the compacted chromosome to be expressed and replicated, unwinding or relaxation of the molecule is required.

In contrast to the bacterial chromosome, the chromosomes of parasites and fungi number more than one per cell, are linear, and are housed within a membrane-bound organelle (the nucleus) of the cell. This difference is a major criterion for classifying bacteria as prokaryotic organisms and fungi and parasites as eukaryotes. The genome topology of a virus may consist of DNA or RNA contained within a protein coat rather than a cell.

Nonchromosomal Elements of the Genome

Although the bacterial chromosome represents the majority of the genome, not all genes in a given cell are confined to the chromosome. Many genes may also be located on plasmids and transposable elements. Both of these extrachromosomal elements are able to replicate and encode information for the production of various cellular products. Although considered part of the bacterial genome, they are not as stable as the chromosome and may be lost during cellular replication, often without any detrimental effects on the viability of the cell.

Plasmids exist as double-stranded, closed, circular, autonomously replicating extrachroosomal genetic elements ranging in size from 1 to 2 kilobases up to 1 megabase or more. The number of plasmids per bacterial cell varies extensively, and each plasmid is composed of several genes. Some genes encode products that mediate plasmid replication and transfer between bacterial cells, whereas others encode products that provide a specialized function, such as determinants of antimicrobial resistance or a unique metabolic process. Unlike most chromosomal genes, plasmid genes do not usually encode for products essential for viability. Plasmids, in whole or in part, may also become incorporated into the chromosome.

Transposable elements are pieces of DNA that move from one genetic element to another, from plasmid to chromosome or vice versa. Unlike plasmids, they are unable to replicate independently and do not exist as separate entities in the bacterial cell. The two types of transposable elements are the simple transposon or insertion sequence (IS) and the composite transposon. Insertion sequences are limited to containing the genes that encode information required for movement from one site in the genome to another. Composite transposons are a cassette (grouping of genes) flanked by insertion sequences. The internal gene imbedded in the insertion sequence encodes for an accessory function, such as antimicrobial resistance. Plasmids and transposable elements coexist with chromosomes in the cells of many bacterial species. These extrachromosomal elements play a key role in the exchange of genetic material throughout the bacterial microbiosphere, including genetic exchange among clinically relevant bacteria.

Expression of Genetic Information

Gene expression is the processing of information encoded in genetic elements (i.e., chromosomes, plasmids, and transposons), which results in the production of biochemical molecules, including RNA molecules and proteins. The overall process of gene expression is composed of two complex steps, transcription and translation. Gene expression requires various components, including a DNA template representing a single gene or cluster of genes, various enzymes and cofactors, and RNA molecules of specific structure and function.

Transcription.

Gene expression begins with transcription. During transcription the DNA base sequence of the gene (i.e., the genetic code) is converted into an mRNA molecule that is complementary to the gene’s DNA sequence (Figure 2-5). Usually only one of the two DNA strands (the sense strand) encodes for a functional gene product. This same strand is the template for mRNA synthesis.

RNA polymerase is the enzyme central to the transcription process. The enzyme is composed of four protein subunits and a sigma factor. Sigma factors are required for the RNA polymerase to identify the appropriate site on the DNA template where transcription of mRNA is initiated. This initiation site is also known as the promoter sequence. The remainder of the enzyme functions to unwind the double-stranded DNA at the promoter sequence and use the DNA strand as a template to sequentially add ribonucleotides (ATP, GTP, uracil triphosphate [UTP], and CTP) to form the growing mRNA strand.

Transcription proceeds in a 5’ to 3’ direction. However, in mRNA, the TTP of DNA is replaced with UTP. TTP contains thymine, and UTP contains uracil. Both molecules contain a heterocyclic ring and are classified as pyrimidines. During synthesis and modification of these molecules, a portion of the molecules are dehydroxylated, forming a 2′-deoxy-nucleotide monophosphate. The dUMP (dehydroxylated uracil monophosphate) is then methylated, forming dTMP (dehydroxylated thymine monophosphate). Following phosphorylation, thymine is only found in the final state as deoxythymidine and therefore cannot be incorporated into an RNA molecule. Synthesis of the single-stranded mRNA product ends when specific nucleotide base sequences on the DNA template are encountered. Termination of transcription may be facilitated by a rho (a prokaryotic protein) cofactor or an intrinsic termination sequence. Both of these mechanisms disrupt the mRNA-RNA polymerase template DNA complex.

In bacteria, the mRNA molecules that result from the transcription process are polycistronic, that is, they encode for several gene products. Frequently, polycistronic mRNA may encode several genes whose products (proteins) are involved in a single or closely related cellular function. When a cluster of genes is under the control of a single promoter sequence, the gene group is referred to as an operon.

The transcription process not only produces mRNA but also tRNA and rRNA. All three types of RNA have key roles in protein synthesis.