Molecular Organization of Bacterial Genome

83

Regulation of Gene Expression

Genetic information is encoded in a sequence of deoxyribonucleic acid (DNA) nucleotides. RNA viruses contain genetic information in ribonucleic acid (RNA) sequence.

DNA molecule is folded as the double-stranded helix, where complementary bases (A-T, G-C) are linked by hydrogen bonds. The bases are coupled to deoxyribose-phosphate resulting in nucleotide formation. Supercoiling of nucleic acid chains provides compact DNA structure in vivo conditions. The size of a DNA molecule is usually expressed in thousands of base pairs (kilobase pairs, kbp). The size of genome of smallest viruses doesn’t exceed 5 kbp, whereas bacterial DNA molecule of nucleoid comes to 5000 kbp (Escherichia coli genome is about 4600 kbp).

Double-stranded DNA is reproduced by semiconservative replication. Each maternal strand serves as the template for newly synthesized strand of DNA, and the replication fork is formed. After replication each daughter DNA molecule contains one maternal strand and one newly formed strand.

Genome of bacteria contains circular DNA of bacterial chromosome (nucleoid).

Genomic ribonucleic acid (RNA) of viruses can exist both in single-stranded or double-stranded form. The base uracil (U) in RNA plays the same role as thymine (T) in DNA for hydrogen bonds formation, and complementary base pairs look like A-U and C-G. RNA is the source of genetic information in RNA-genomic viruses.

Genetic code is realized into final protein sequence via messenger RNA (mRNA). Usually RNA polymerase forms a single polyribonucleotide strand of messenger RNA (mRNA), using DNA as a template. This process is called transcription. The mRNA attains a nucleotide sequence complementary to the template DNA strand. In bacteria the newly formed mRNA is polycistronic, containing the information about the group of related proteins encoded by several genes.

The ribosomes composed of ribosomal RNA (rRNA) and proteins transfer the genetic information from mRNA into the primary structure of proteins by the action of aminoacyl-transfer RNAs (tRNAs). The latter process is known as translation.

Regulation of expression of bacterial genes is quite complex and intricate.

Genes in bacterial genome can be organized into operon clusters. Operon is a structural and functional unit of bacterial genomic organization. It encodes and therefore controls a set of related structural proteins and chemical reactions.

Operon usually comprises promoter, operator, structural genes and in many cases regulatory gene.

Specific regulatory proteins (e.g., repressors) encoded by regulatory genes influence the expression of structural genes.

Structural genes predominantly encode enzymes and structural proteins. DNA transcription resulting in polycistronic mRNA synthesis is initiated from the promoter site – a specific sequence of DNA capable of binding RNA polymerase for transcription initiation.

Regulatory proteins that attach to the regions of DNA nearby promoters also actively participate in expression of structural genes. Short DNA sequence between promoter site and structural genes known as operator binds to regulatory proteins (e.g., repressors) to control transcription.

Inhibition of transcription by repressor proteins is termed as negative control. The opposite case – initiation of transcription by binding of so-called activator proteins to bacterial DNA is termed as positive control.

A single regulatory gene governs the transcription of several structural genes. For instance, five genes affecting tryptophan biosynthesis are clustered within the trp operon in E. coli.

Trp operon of E. coli is regarded as negative respressible operon in bacteria. In this case the genetic expression is controlled by repression mechanism.

For trp operon of E. coli it functions as follows:

if tryptophan amino acid is sufficiently present in the medium and thereafter within bacterial cell, it leads to tryptophan binding to the repressor protein. This results in changes of repressor conformation ensuring repressor protein binding to DNA sequence of trp operator. This binding of the repressor blocks the transcription of structural trp genes responsible for tryptophan synthesis.

In the opposite case of tryptophan lack in the medium, the repressor protein doesn’t block operator sequence, and the expression of structural genes starts, resulting in the synthesis of tryptophan necessary for bacterial cell growth.

The variations of bacterial negative inducible operons are also well-presented in bacterial genomes.

For instance, E. coli harbors the lac operon, responsible for lactose metabolism in bacteria.

This operon carries three structural genes. Among them, the lacY gene governs the transport of lactose across the membrane into the bacterial cell. The lacZ gene codes for beta-galactosidase, the enzyme that hydrolyzes lactose resulting in production of galactose and glucose. Glucose is further used by bacterial cell in the pathways of energy metabolism. In addition, some limited amounts of lactose can be converted by beta-galactosidase into its isomer allolactose.

In case of lactose absence the lac operon is almost silent and only the trace amounts of its proteins (e.g., galactosidase) can be expressed.

This is achieved by binding the repressor protein (encoded by regulatory gene) to operator DNA sequence thereby blocking transcription of structural genes (negative transcriptional control).

When the lactose concentration arises, the initially present beta-galactosidase produces some amounts of allolactose from available lactose. Allolactose is the direct inducer of the lac operon. It binds to the repressor, causing the dissociation of repressor-operator complex. This in turn makes available the promoter site for RNA polymerase that begins the transcription of structural genes. Structural genes products (e.g., galactosidase enzyme) utilize lactose for cellullar needs.

In addition, the expression of many operons is directly stimulated by the transcriptional activator proteins. For the regulation of transcription they interact with a specific DNA fragment called enhancer sequence.

The enhancer sequence is located near the promoter site within the regulated operon. Activator proteins ensure positive transcriptional control stimulating RNA polymerase activity.

As an example, E. coli expresses regulatory molecules of cyclic AMP-binding protein (CAP). It is stimulated by specific cellular metabolite 3′,5′-cyclic AMP (or cAMP). This substance (cAMP), shown to arise in energy-exhausted cells, activates the CAP thus enhancing the expression of catabolic enzymes that elevate the yield of metabolic energy.