Which of the following transcriptional control features are found in both prokaryotes and eukaryotes?

Introduction

Prokaryotic cells such as E. coli can regulate their gene expression to adapt to the changes in available nutrients in the surrounding environment. Their regulation of gene expression occurs mainly through regulation of the rate of mRNA synthesis, since their transcription and translation is coupled. For example, in a lactose rich medium, Escherichia coli activates expression of the gene clusters called operons that are required for the processing of lactose, e.g., β-galactosidase, permease, etc. However, eukaryotic multicellular organisms regulate their gene expression through five major levels: modification of chromatin structures, the number of mRNA molecules synthesized [transcriptional], processing of the mRNA [post-transcriptional], the rate of mRNA translation [translational], and modifications of the proteins for proper function [post-translational regulation]. Eukaryotic regulation of gene expression is thus much more complex than prokaryotic gene expression and involves a variety of factors and elements. In this chapter, the numerous mechanisms and factors by which regulation of gene expression is achieved in prokaryotes and eukaryotes are described.

I The Diversity of Prokaryotic Organisms

Prokaryotic cells represent the smallest and simplest form of life that can metabolize, grow and reproduce. They are presumed to resemble the earliest forms of life and they reproduce much more quickly than multicellular organisms do. Taken together, these two properties imply that prokaryotic organisms have had more opportunity to evolve [by orders of magnitude] than plants or animals have had; accordingly, this predicts that prokaryotes should have the most functionally efficient, diverse and specialized of cells, despite their structural simplicity. The key to this paradox is the recognition that genetic variation and natural selection should allow a unicellular organism to improve its performance and acquire new functions without becoming structurally complex. Furthermore, as environments, survival strategies and ecological niches change over time, the criteria of optimal cellular function change which, in turn, sets the stage for new rounds of optimization in various directions. The resulting diversification and specialization can also be expected to make certain features superfluous in certain lineages, leading to cells that may be even simpler than their predecessor.

Functional specialization and optimization of a structurally simple cell seems to account for the observed diversity of modern prokaryotes. Molecular measures of divergence, such as small-subunit ribosomal RNA sequence, indicate that two prokaryotic lineages separated very early and that each encompasses more molecular diversity than multicellular organisms [Fig. 50.1]. The two groups distinguished by this early split, Bacteria and Archaea, each have phylogenetic status equivalent to that of all eukaryotic organisms and the three resulting taxonomic units have been termed Domains [Woese et al., 1990]. This extensive divergence is also evident in terms of cellular function. Certain bacteria and archaea have metabolic properties not represented among eukaryotes, including N2 fixation, anoxic photosynthesis, additional routes of CO2 assimilation and adaptation to extreme environmental conditions. Similarly, archaea [singular: archaeon, or less commonly, archaeum] have cellular features and metabolic pathways not found in bacteria. These uniquely archaeal features include isoprenoid membrane lipids [found in all archaea] and the ability to make methane [found in a number of genera].

One practical consequence of the deep diversity of the bacterial and archaeal lineages is that it precludes any one organism, such as the bacterium Escherichia coli, from modeling all aspects of prokaryotic physiology, even though this and several other species can be analyzed in great detail. It should also be noted that some components of eukaryotic cells have bacterial origins. In particular, at least two eukaryotic organelles, the mitochondrion and the chloroplast, resulted from endosymbiotic acquisitions of bacteria by progenitors of modern eukaryotic cells [Scwartz and Dayhoff, 1978]. This relationship provides a context for understanding molecular structure and function of both the eukaryotic organelle and the bacterial cell.

CELL WALLS

Prokaryotic cells are surrounded by a generally rigid cell wall, protecting the cell from osmotic lysis [Figs. 5.5 and 5.6]. The cell wall has also been important in identification and classification of bacteria, providing a major division between gram-positive and gram-negative bacteria, defined on the basis of the Gram stain. The cell wall of gram-positive cells consists of a single thick layer of peptidoglycan, surrounding the cytoplasmic membrane. Peptidoglycan is a polymer consisting of a backbone of alternating N-acetylglucosamine and N-acetylmuramic acid residues connected to cross-linked peptide chains of four amino acids. Gram-positive cell walls also usually contain teichoic acids, polymers of glycerol or ribitol, linked by phosphate groups and containing amino acids and sugars. In the more complex gram-negative cell wall, the peptidoglycan layer is much thinner and is surrounded by an outer membrane enclosing a periplasmic space, which contains enzymes involved in nutrient acquisition, electron transport, and protection from toxins. In contrast, archaeal cell walls have variable chemical structure, consisting of proteins, glycoproteins, or polysaccharides, but do not contain peptidoglycan [Fig. 5.6].

The cell walls of many bacteria are encased within extracellular material [Fig. 5.5], ranging from apparently rigid and distinct capsules of specific thickness to more diffuse [chemically and physically] extracellular polymeric substances. Many roles have been assigned to this material, including protection from predation, adhesion to solid surfaces, and biofilm formation. In the free-living N2-fixing bacterium Azotobacter, extracellular material is important in creating anaerobic regions required for N2 fixation. Biofilm formation is particularly important, with suggestions that the majority of the soil microbial community is attached to particulate matter [clay minerals, soil organic matter, plant roots, and animals]. Particulate material provides a concentration of nutrients necessary for microbial growth, and surface attachment has been shown to increase survival of bacteria and to protect them from environmental stress, including low pH, starvation, and inhibition by antibiotics and heavy metals. An example is the production by nitrifying bacteria, in model soil systems, of copious amounts of extracellular material that effectively forms a blanket over colonies, such that individual cells are not visible. This occurs despite the fact that these autotrophic organisms gain barely sufficient energy from oxidation of ammonium or nitrite, use much of this energy to generate reducing equivalents, and require more reducing equivalents because of the requirement to fix CO2. However, once formed, biofilms of these organisms are protected from a wide range of factors to which suspended cells are susceptible. Attachment of cells to surfaces is also facilitated by short, hair-like fimbriae, while similar structures, sex pili, are involved in cell–cell contact associated with plasmid transfer.

C Cell Walls

Prokaryotic cells are surrounded by a rigid cell wall, protecting the cell from osmotic lysis [Figs. 3.5 and 3.6]. The cell wall is important in diagnosis and phenotypic classification of bacteria, providing a major division between Gr+ and Gr− bacteria. The cell wall of Gr+ cells consists of a single layer of peptidoglycan, surrounding the cytoplasmic membrane. Peptidoglycan is a polymer consisting of a backbone of alternative N-acetylglucosamine and N-acetylmuramic acid residues connected to cross-linked peptide chains of four amino acids. Gram-positive cell walls also usually contain teichoic acids, polymers of glycerol or ribitol, linked by phosphate groups, and containing amino acids and sugars. In the more complex Gr+ cell wall, the peptidoglycan layer is much thinner and is surrounded by an outer membrane enclosing a periplasmic space, which contains enzymes involved in nutrient acquisition, electron transport, and protection from toxins.

Archaeal cell walls differ from those of bacteria and provide a major exception to the original definition of prokaryotes. They lack an outer membrane, and peptidoglycan is replaced by a range of compounds, including pseudomurein [similar to peptidoglycan] or other polysaccharides. Many archaeal cell walls contain paracrystalline protein or glycoprotein, the S-layer, which is also found in some bacteria. In addition, some prokaryotes do not possess a cell wall [e.g., intracellular mycoplasmas and the archaeon Thermoplasma, which have strengthened membranes].

The cell walls of many bacteria are encased within extracellular material [Fig. 3.5], ranging from apparently rigid and distinct capsules of specific thickness to more diffuse [chemically and physically] extracellular polymeric substances. Many roles have been assigned to this material, including protection from predation, adhesion to solid surfaces, and biofilm formation. In the free-living, N2-fixing bacterium Azotobacter [a member of the Pseudomonadales], limited O2 diffusion through extracellular material creates anaerobic regions required for N2 fixation. Biofilm formation is particularly important, with suggestions that the majority of the soil microbial community is attached to particulate matter [clay minerals, soil organic matter [SOM], plant roots, and animals]. Adsorption of nutrients to particulate material provides concentrations necessary for microbial growth. Surface attachment can increase survival of bacteria and protect them from environmental stress, including low pH, starvation, and inhibition by antibiotics and heavy metals. An example is the production by nitrifying bacteria in soil model systems of copious amounts of extracellular material that effectively form a blanket over colonies. This occurs despite the fact that these autotrophic organisms barely gain sufficient energy from oxidation of ammonia or nitrite, use much of this energy to generate reducing equivalents, and then require more reducing equivalents because of the requirement to fix CO2. However, once formed, biofilms of these organisms are protected from a wide range of factors to which suspended cells are susceptible [Prosser, 2011]. Attachment of cells to surfaces is also facilitated by short, hair-like fimbriae, whereas similar structures, sex pili, are involved in cell-to-cell contact associated with plasmid transfer.

Normal Physiological Processes

Prokaryotic cells derive their energy from glycolysis, a process that oxidizes and splits the six-carbon glucose molecule into two three-carbon pyruvate molecules [Figure 1]. The energy released during glycolysis is conserved by the phosphorylation of adenosine diphosphate [ADP] to adenosine triphosphate [ATP] and by the reduction of nicotinamide adenine dinucleotide [NAD+] to NADH. Glycolysis would stop if it lacks supply of NAD+. Higher organisms regenerate NAD+ by reducing pyruvate to lactate, allowing glycolysis to continue indefinitely:

Glucose+2ADP+2Pi→2lactate+ 2ATP

where Pi denotes inorganic phosphate.

The hydrolysis of ATP provides the energy required to power vital cellular processes, such as protein synthesis, locomotion, and membrane-associated ionic pumps:

2ATP→2 ADP+2Pi+2H++energy

Combining the previous equations yields the general expression for glycolysis:

Glucose→2lactate+2H++energy

The emergence of mitochondria, remarkable organelles capable of handling oxygen safely, provided the energy required for the evolution of eukaryotic cells. Mitochondria are thought to be descendants from symbiotic bacteria since they contain their own DNA and replicate independently.

Mitochondria generate vast quantities of energy from O2, generating CO2 and water as by-products. In the mitochondria, pyruvate is oxidized to acetyl-CoA, which enters the tricarboxylic acid cycle, producing CO2 and electrons. These electrons move down a cascade of progressively lower energy states until transferred to oxygen, the final electron acceptor. The energy liberated by the mitochondrial electron cascade is coupled to the partly understood mechanism of oxidative phosphorylation, producing 36 ATP molecules per glucose molecule.

The abundant cellular energy provided by aerobic metabolism allowed eukaryotic cells to form complex multicellular organisms. Growth, however, was limited by the diffusion of O2 from the environment. Species adapted to prevailing ecological conditions by developing organs to carry O2 from the surrounding media to all tissue cells. Animal O2 transport systems consist of heme pigments to reversibly bind O2 [hemoglobin], specialized cells to enfold but not consume the potentially toxic O2 [erythrocytes], organs to promote O2 diffusion from the environment into blood [lungs or gills], and the hydraulic pumps and circulatory conduits needed to convey O2-containing cells to and from the tissues [the heart and blood vessels].

Air reaches the alveolar spaces following a negative pressure gradient during inspiration. Alveolar O2 partial pressure [PAO2] may be estimated from the alveolar air equation

PAO2=FIO2[Patm−Pwater]−PaCO2[FIO2+[1−FIO2]/RQ] mmHg

where FIO2 is the inspired O2 fraction [0.21 for air], Patm is the atmospheric pressure, Pwater is the water vapor pressure, PaCO2 is the arterial blood CO2 partial pressure, and RQ is the respiratory quotient.

When breathing air at sea level, this equation approximates to

PAO2=150−PaCO2/0.8mmHg

Oxygen diffuses from the alveoli into pulmonary capillary blood, where it dissolves in plasma or binds to hemoglobin. The alveolar–arterial gradient is the difference between alveolar and arterial PO2 [A−a=PAO2−PaO2]. Ventilation perfusion inequality, shunt, or [rarely] diffusion limitation of oxygen can widen the A–a gradient across the blood–gas barrier.

The sigmoid-shaped oxyhemoglobin dissociation curve [ODC] defines the relationship between plasma PO2 and fractional hemoglobin O2 saturation [SO2]. The concentration or O2 content of blood is the sum of O2 bound to hemoglobin and O2 dissolved in plasma:

O2content=13.9×SO2×[Hb]+0.031 ×PO2mlO2perliterof blood

where the term 13.9 is the O2 carrying capacity of hemoglobin, [Hb] is the concentration of hemoglobin in blood [g l−1], and the term 0.031 is the solubility of O2 in plasma for PO2 in mmHg.

Arterial blood delivers oxygen and metabolic substrate to the tissues in accordance to local energy requirements. Higher level control of the circulation is aimed primarily at counteracting gravitational effects by stimulation of arteriolar α and β receptors. The microvasculature – the vast array of terminal arterioles, capillaries, and venules – regulates intraorgan blood flow through vasomotion, rhythmic oscillations in vascular tone.

Accurate measurements of regional O2 delivery are difficult to obtain. A first approximation to the adequacy of cellular O2 transport may be obtained by calculating the systemic rates of O2 delivery [DO2] and O2 uptake [VO2],

DO2=cardiacoutput×O2contentmlO2perminute

Systemic O2 uptake [VO2] may be measured directly from the expired gases as

VO2=[FIO2[1−FEO2−FECO2]/[1−FIO2]−FEO2]×VEmlO2perminute

where FIO2 and FEO2 denote the inspired and expired fractional O2 concentration, respectively; FECO2 is the fractional concentration of expired CO2; and VE is the expired minute volume. A practical problem with this expression is that small inaccuracies in measuring FIO2 at values >0.60 may produce large errors in VO2.

Systemic VO2 may also be calculated with the aid of Fick's principle as

VO2=cardiacoutput×[arterial O2content−mixedvenousO2content ]mlO2perminute

This calculation requires the insertion of a pulmonary artery catheter to gain access to pulmonary artery blood. Fick's principle underestimates total VO2 since it does not account for pulmonary VO2. The latter can be substantial in severe pneumonia or in adult respiratory distress syndrome.

The ratio of tissue CO2 production [VCO2] to VO2 is the respiratory quotient,

RQ=V CO2/VO2

The type of substrate consumed determines the value of RQ, being 0.7 for free fatty acids and 1.0 for glucose.

The O2 extraction ratio [ERO2], the fraction of DO2 taken up by the tissues, is another useful parameter:

ERO2=VO2/DO2

ERO2 is approximately 20–25% at rest, increasing to 50% or greater with exercise or during cardiac failure at rest. ERO2 values