Citation:Griswold,A.(2008)Genome packaging in prokaryotes: the circular chromosome of E. coli.occupychristmas.org Education1(1):57
How do bacteria, lacking a nucleus, organize and pack their genome into the cell? Supercoiling enables this but forces a different kind of transcription and translation in prokaryotes.
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Most students learnat an early age that organisms can be broadly divided into two types: prokaryotesand eukaryotes. In primary school, children are taught that the main differencebetween these organisms is that eukaryotic cells contain membrane-boundorganelles, such as the nucleus, while prokaryotic cells do not. There is muchmore to the story, however, particularly with regard to chromosomal structureand organization.
Much of what isknown about prokaryotic chromosome structure was derived from studies of Escherichia coli, a bacterium that livesin the human colon and is commonly used in laboratory cloning experiments. Inthe 1950s and 1960s, this bacterium became the model organism of choice for prokaryoticresearch when a group of scientists used phase-contrast microscopy andautoradiography to show that the essential genes of E. coli are encoded on a single circular chromosome packaged withinthe cell nucleoid (Mason & Powelson, 1956; Cairns, 1963).
Prokaryotic cells donot contain nuclei or other membrane-bound organelles. In fact, the word “prokaryote”literally means “before the nucleus.” The nucleoid is simply the area of aprokaryotic cell in which the chromosomal DNA is located. This arrangement isnot as simple as it sounds, however, especially considering that the E. coli chromosome is several orders ofmagnitude larger than the cell itself. So, if bacterial chromosomes are sohuge, how can they fit comfortably inside a cell—much less in one small cornerof the cell?
The answer to this question lies in DNA packaging. Whereas eukaryotes wrap their DNA around proteins called histones to help package the DNA into smaller spaces, most prokaryotes do not have histones (with the exception of those species in the domain Archaea). Thus, one way prokaryotes compress their DNA into smaller spaces is through supercoiling (Figure 1). Imagine twisting a rubber band so that it forms tiny coils. Now twist it even further, so that the original coils fold over one another and form a condensed ball. When this type of twisting happens to a bacterial genome, it is known as supercoiling. Genomes can be negatively supercoiled, meaning that the DNA is twisted in the opposite direction of the double helix, or positively supercoiled, meaning that the DNA is twisted in the same direction as the double helix. Most bacterial genomes are negatively supercoiled during normal growth.
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During the 1980s and1990s, researchers discovered that multiple proteins act together to fold andcondense prokaryotic DNA. In particular, one protein called HU, which is themost abundant protein in the nucleoid, works with an enzyme called topoisomeraseI to bind DNA and introduce sharp bends in the chromosome, generating thetension necessary for negative supercoiling. Recent studies have also shownthat other proteins, including integration host factor (IHF), can bind tospecific sequences within the genome and introduce additional bends (Rice et al., 1996). The folded DNA is thenorganized into a variety of conformations (Sinden & Pettijohn, 1981) thatare supercoiled and wound around tetramers of the HU protein, much likeeukaryotic chromosomes are wrapped around histones (Murphy & Zimmerman,1997).
Once the prokaryoticgenome has been condensed, DNA topoisomerase I, DNA gyrase, and other proteinshelp maintain the supercoils. One of these maintenance proteins, H-NS, plays anactive role in transcription by modulating the expression of the genes involvedin the response to environmental stimuli. Another maintenance protein, factorfor inversion stimulation (FIS), is abundant during exponential growth andregulates the expression of more than 231 genes, including DNA topoisomerase I(Bradley et al., 2007).
Supercoilingexplains how chromosomes fit into a small corner of the cell, but how do theproteins involved in replication and transcription access the thousands ofgenes in prokaryotic chromosomes when everything is packaged together sotightly? It has been determined that prokaryotic DNA replication occurs at arate of 1,000 nucleotides per second, and prokaryotic transcription occurs at arate of about 40 nucleotides per second (Lewin, 2007), so bacteria must havehighly efficient methods of accessing their DNA strands. But how?
Researchers havenoted that the nucleoid usually appears as an irregularly shaped mass withinthe prokaryotic cell, but it becomes spherical when the cell is treated withchemicals to inhibit transcription or translation. Moreover, duringtranscription, small regions of the chromosome can be seen to project from thenucleoid into the cytoplasm (i.e., the interior of the cell), where they unwindand associate with ribosomes, thus allowing easy access by varioustranscriptional proteins (Dürrenberger et al., 1988). These projectionsare thought to explain the mysterious shape of nucleoids during active growth.When transcription is inhibited, however, the projections retreat into thenucleoid, forming the aforementioned spherical shape.
Because there is nonuclear membrane to separate prokaryotic DNA from the ribosomes within thecytoplasm, transcriptionand translation occur simultaneously in these organisms. This is strikinglydifferent from eukaryotic chromosomes, which are confined to the membrane-boundnucleus during most of the cell cycle. In eukaryotes, transcription must becompleted in the nucleus before the newly synthesized mRNA molecules can be transportedto the cytoplasm to undergo translation into proteins.
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Recently, it hasbecome apparent that one size does not fit all when it comes to prokaryoticchromosome structure. While most prokaryotes, like E. coli, contain a single circular DNA molecule that makes up theirentire genome, recent studies have indicated that some prokaryotes contain asmany as four linear or circular chromosomes. For example, Vibrio cholerae, the bacteria that causes cholera, contains twocircular chromosomes. One of these chromosomes contains the genes involved inmetabolism and virulence, while the other contains the remaining essentialgenes (Trucksis et al., 1998). Aneven more extreme example is provided by Borreliaburgdorferi, the bacterium that causes Lyme disease. This organism istransmitted through the bite of deer ticks (Figure 2), and it contains up to 11 copies of asingle linear chromosome (Ferdows &Barbour, 1989). Unlike E. coli,Borrelia cannot supercoil its linearchromosomes into a tight ball within the nucleoid; rather, these strands arediffused throughout the cell (Hinnebusch& Bendich, 1997).
Other organisms,such as Bacillus subtilis, formnucleoids that closely resemble those of E.coli, but they use different architectural proteins to do so. Furthermore,the DNA molecules of Archaea, a taxonomic domain composed of single-celled,nonbacterial prokaryotes that share many similarities with eukaryotes, can benegatively supercoiled, positively supercoiled, or not supercoiled at all. It isimportant to note that archaeans are the only group of prokaryotes that useeukaryote-like histones, rather than the architectural proteins describedabove, to condense their DNA molecules (Sandman et al., 1990). The acquisition of histones by archaeans is thoughtto have paved the way for the evolution of larger and more complex eukaryoticcells (Minsky et al., 1997).
Most prokaryotesreproduce asexually and are haploid, meaning that only a single copy of eachgene is present. This makes it relatively easy to generate mutations in the laband study the resulting phenotypes. By contrast, eukaryotes that reproducesexually generally contain multiple chromosomes and are said to be diploid,because two copies of each gene exist—with one copy coming from each of an organism”sparents.
Yet anotherdifference between prokaryotes and eukaryotes is that prokaryotic cells oftencontain one or more plasmids (i.e., extrachromosomal DNA molecules that areeither linear or circular). These pieces of DNA differ from chromosomes in thatthey are typically smaller and encode nonessential genes, such as those thataid growth in specific conditions or encode antibiotic resistance. Borrelia, for instance, contains morethan 20 circular and linear plasmids that encode genes responsible forinfecting ticks and humans (Fraser et al.,1997). Plasmids are often much smaller than chromosomes (i.e., less than 1,500kilobases), and they replicate independently of the rest of the genome.However, some plasmids are capable of integrating into chromosomes or movingfrom cell to cell.
Perhaps due to thespace constraints of packing so many essential genes onto a single chromosome,prokaryotes can be highly efficient in terms of genomic organization. Verylittle space is left between prokaryotic genes. As a result, noncodingsequences account for an average of 12% of the prokaryotic genome, as opposedto upwards of 98% of the genetic material in eukaryotes (Ahnert et al., 2008). Furthermore, unlikeeukaryotic chromosomes, most prokaryotic genomes are organized into polycistronicoperons, or clusters of more than one coding region attached to a singlepromoter,separated by only a few base pairs. The proteins encoded by each operon oftencollaborate on a single task, such as the metabolism of a sugar into by-productsthat can be used for energy (Figure 3).
Three structural genes code for proteins involved in lactose import and metabolism in bacteria. The genes are organized together in a cluster called the lac operon.
lac operon.”, “Figure 3”, “Three structural genes code for proteins involved in lactose import and metabolism in bacteria. The genes are organized together in a cluster called the lac operon.”, “627”,”http://www.occupychristmas.org/occupychristmas.org_education”, “The lac operon in bacteria includes a promoter, an operator, and three structural genes. These regions occur in a specific arrangement, and, in the diagram, the operon is presented in order from left to right. The following regions are shown: first the promoter in red, then the operator in yellow, and finally the three structural genes, which include beta-galactosidase in blue, beta-galactoside permease in dark pink, and beta-galactoside transacetylase in orange. Additional regulatory sequences are located to the left of the promoter in the region, upstream of the lac operon.”)” class=”inlineLinks”> Figure Detail
The organization ofprokaryotic DNA therefore differs from that of eukaryotes in several importantways. The most notable difference is the condensation process that prokaryoticDNA molecules undergo in order to fit inside relatively small cells. Otherdifferences, while not as dramatic, are summarized in Table 1.
Table 1: Prokaryotic versusEukaryotic Chromosomes
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