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I have been learning about the chemical structure of RNA, but I can't understand why all hydrogen atoms disappear in the phosophodiester bond between the riboses of RNA.
Phosphoric acid is $H_3PO_4$:
However, when it's connected to ribose the hydrogens disappear:
Where did the hydrogens go and why?
Generally hydrogen is not shown in DNA structure. I have made a simple diagram to show where all hydrogens go:
There are 3 hydrogens in phosphoric acid, each of them leaves as:
1 H joins with 3' -OH of previous nucleotide to form H2O during DNA replication.
1 OH joins with H of next nucleotide to form H2O during DNA replication.
1 H is released as H+ (remember it was phosphoric acid), hence giving rise to the acidic character of DNA (remember it is DeoxyriboNucleic Acid).
I think you should first study DNA replication (if you haven't, yet) to get more thorough knowledge about the topic.
A phosphodiester bond occurs when exactly two of the hydroxyl groups in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. An example is found in the linking of two pentose (5 carbon sugar) rings to a phosphate group by strong, covalent ester bonds. Each ester bond is formed by a condensation reaction in which water is lost. This bond is a key structural feature of the backbone of DNA and RNA and links the 3’ carbon of one nucleotide to the 5’ carbon of another to produce the strands of DNA and RNA.
How is a phosphodiester bond formed?
Like peptide bonds in proteins and glycosidic bonds between monosaccharides, phosphodiester bonds result from dehydration reactions in which a water molecule is lost. Here is the general scheme of one of these dehydration reactions:
Phosphate ions correspond to the completely deprotonated conjugate base of phosphoric acid and are called inorganic phosphates, whose abbreviation is denoted Pi. When two phosphate groups are linked together, an anhydrous phosphate bond forms, and a molecule known as inorganic pyrophosphate or PPi is obtained.
When a phosphate ion is attached to a carbon atom in an organic molecule, the chemical bond is called a phosphate ester, and the resulting species is an organic monophosphate. If the organic molecule binds to more than one phosphate group, organic diphosphates or triphosphates are formed.
When a single inorganic phosphate molecule is attached to two organic groups, a phosphodiester or "phosphate diester" bond is employed. It is important not to confuse phosphodiester bonds with high-energy phosphoanhydro bonds between the phosphate groups of molecules like ATP, for example.
The phosphodiester linkages between adjacent nucleotides consist of two phosphoester linkages that occur between the hydroxyl at the 5 'position of one nucleotide and the hydroxyl at the 3' position of the next nucleotide on a DNA or RNA strand.
Depending on the conditions of the environment, these bonds can be hydrolyzed both enzymatically and non-enzymatically.
Harnessing RNAi to Silence Viral Gene Expression
188.8.131.52 Optimizing siRNA Stability
Because phosphodiester bond cleavage and resultant RNA breakdown typically occurs by a transesterification reaction involving the 2′-hydroxyl group of the ribose, chemical modification of this moiety within siRNAs has been a major method of improving their stability. Alterations to the phosphodiester backbone and substituting ribose for alternative sugars have also been used to extend the half-lives of siRNAs. Ribose modifications involving the 2′ -hydroxyl include incorporation of 2′- O-methyl (2′OMe), 2′-fluoro (2′-F), locked nucleic acids (LNAs), acyclic unlocked nucleic acids (UNAs), 2′-O-methoxyethyl (2′MOE), guanidinopropyl (GP), and deoxy residues. Backbone alterations to siRNAs have involved the use of phosphorothioate and boranophosphate residues  , both of which have been reported to improve siRNA stability and silencing efficacy. Variable numbers of nucleotides may be modified within a siRNA and different chemical modifications may be added to single siRNAs. When formulated within protective nonviral vectors, chemical modification to protect siRNAs may not be as important as when these silencers are administered naked. In addition to improving siRNA stability, chemical modification may be used to influence siRNA duplex stability, target interaction, and immunostimulation.
The most widely studied ribose alteration is the 2′OMe substitution  . This nucleotide variant occurs naturally and is not toxic. Several studies have shown that siRNAs containing 2′OMe residues improve siRNA stability while retaining silencing efficacy  . The modifications may enhance resistance to both exo- and endonucleases. Positioning 2′OMe residues at different nucleotides of the siRNAs, alone or in combination, has been found to limit degradation. For example, strands with 2′OMe in nucleotides at the 3′ or 5′ ends of the guide strand, all nucleotides of the passenger strand, and at alternating ribose moieties of the guide may all contribute some improvement to stability  . Another approach entailing use of 2′OMe modification to stabilize siRNAs is particularly important for retaining guide strand efficacy. It is established that the lower thermodynamic stability at the 5′ end of a strand is important for biasing strand selection  . However, this property may also make the intended oligonucleotide more vulnerable to exonucleases with 5′ to 3′ activity that are found in the blood  . Incorporation of 2′OMe residues at the 5′ end of the guide has been shown to be useful to provide resistance to this degradation. UNAs have also been used successfully to confer nuclease resistance on siRNAs  . The ribose moieties in UNAs lack the C2–C3 bond, which results in opening of the sugar ring. The acyclic residues are poor substrates for nuclease degradation.
LNAs contain ribose molecules with a bicyclic structure. The linkage between the 2′ oxygen and 4′ carbon atoms results in a 3′-endo configuration, which increases Tm within duplexes and serum stability  . Oligonucleotides with LNA modifications are now in widespread use. However, excessive LNA residues within a siRNA may diminish silencing efficacy and also cause some toxicity  .
DNA and RNA
The two main types of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) . DNA is the genetic material found in all living organisms, ranging from single-celled bacteria to multicellular mammals. It is found in the nucleus of eukaryotes and in the organelles, chloroplasts, and mitochondria. In prokaryotes, the DNA is not enclosed in a membranous envelope.
The entire genetic content of a cell is known as its genome, and the study of genomes is genomics. This includes both the chromosomal and extra-chromosomal DNA. Chromosomes in prokaryotes have very few differences when compared to eukaryotes. These differences are mostly in size, number and shape. Both have packaging proteins, both contain many genes. Eukaryotes generally have more DNA, higher numbers and a linear shape, as compared to circular in prokaryotes. Also in eukaryotic cells, DNA forms a complex with histone proteins to form chromatin, the substance of eukaryotic chromosomes. A chromosome may contain hundreds to tens of thousands of genes. Many genes contain the information to make protein products other genes code for RNA products. DNA controls all of the cellular activities by turning the genes &ldquoon&rdquo or &ldquooff.&rdquo
Another type of nucleic acid, RNA, is mostly involved in protein synthesis. The DNA molecules never leave the nucleus (in eukaryotes) or nucleoid (in prokaryotes) but instead use an intermediary to communicate with the rest of the cell. This intermediary is the messenger RNA (mRNA) . Other types of RNA&mdashlike rRNA, tRNA, and microRNA&mdashare also involved in protein synthesis and its regulation.
DNA and RNA are polymers made up of monomers known as nucleotides . The nucleotides combine can with each other to form a polynucleotide . Each nucleotide is made up of three components: a nitrogenous base, a pentose (five-carbon) sugar, and one or more phosphate groups (Figure (PageIndex<1>)). Each nitrogenous base in a nucleotide is attached to a sugar molecule, which is attached to the phosphate group(s).
The nitrogenous bases, important components of nucleotides, are organic molecules and are so named because they contain carbon and nitrogen. They are bases because they contain an amino group that has the potential of binding an extra hydrogen, and thus, decreases the hydrogen ion concentration in its environment, making it more basic. Each nucleotide in DNA contains one of four possible nitrogenous bases: adenine (A), guanine (G) cytosine (C), and thymine (T).
Adenine and guanine are classified as purines . The primary structure of a purine is two carbon-nitrogen rings. Cytosine, thymine, and uracil are classified as pyrimidines which have a single carbon-nitrogen ring as their primary structure (Figure (PageIndex<1>)). Each of these basic carbon-nitrogen rings has different functional groups attached to it. In molecular biology shorthand, the nitrogenous bases are simply known by their symbols A, T, G, C, and U. DNA contains A, T, G, and C whereas RNA contains A, U, G, and C.
The pentose sugar in DNA is deoxyribose, and in RNA, the sugar is ribose (Figure (PageIndex<1>)). The difference between the sugars is the presence of the hydroxyl group on the second carbon of the ribose and hydrogen on the second carbon of the deoxyribose. The carbon atoms of the sugar molecule are numbered as 1&prime, 2&prime, 3&prime, 4&prime, and 5&prime (1&prime is read as &ldquoone prime&rdquo). The phosphate residue is attached to the hydroxyl group of the 5&prime carbon of one sugar and the hydroxyl group of the 3&prime carbon of the sugar of the next nucleotide, which forms a 5&prime&ndash3&prime phosphodiester linkage. The phosphodiester linkage is not formed by simple dehydration reaction like the other linkages connecting monomers in macromolecules: its formation involves the removal of two phosphate groups. A polynucleotide may have thousands of such phosphodiester linkages.
Figure (PageIndex<1>): A nucleotide is made up of three components: a nitrogenous base, a pentose sugar, and one or more phosphate groups. Carbon residues in the pentose are numbered 1&prime through 5&prime (the prime distinguishes these residues from those in the base, which are numbered without using a prime notation). The base is attached to the 1&prime position of the ribose, and the phosphate is attached to the 5&prime position. When a polynucleotide is formed, the 5&prime phosphate of the incoming nucleotide attaches to the 3&prime hydroxyl group at the end of the growing chain. Two types of pentose are found in nucleotides, deoxyribose (found in DNA) and ribose (found in RNA). Deoxyribose is similar in structure to ribose, but it has an H instead of an OH at the 2&prime position. Bases can be divided into two categories: purines and pyrimidines. Purines have a double ring structure, and pyrimidines have a single ring.
DNA Double-Helix Structure
DNA has a double-helix structure (Figure (PageIndex<2>)). The sugar and phosphate lie on the outside of the helix, forming the backbone of the DNA. The nitrogenous bases are stacked in the interior, like the steps of a staircase, in pairs the pairs are bound to each other by hydrogen bonds. Every base pair in the double helivx is separated from the next base pair by 0.34 nm. The two strands of the helix run in opposite directions, meaning that the 5&prime carbon end of one strand will face the 3&prime carbon end of its matching strand. (This is referred to as antiparallel orientation and is important to DNA replication and in many nucleic acid interactions.)
Figure (PageIndex<2>): Native DNA is an antiparallel double helix. The phosphate backbone (indicated by the curvy lines) is on the outside, and the bases are on the inside. Each base from one strand interacts via hydrogen bonding with a base from the opposing strand. (credit: Jerome Walker/Dennis Myts)
Only certain types of base pairing are allowed. For example, a certain purine can only pair with a certain pyrimidine. This means A can pair with T, and G can pair with C, as shown in Figure (PageIndex<3>). This is known as the base complementary rule. In other words, the DNA strands are complementary to each other. If the sequence of one strand is AATTGGCC, the complementary strand would have the sequence TTAACCGG. During DNA replication, each strand is copied, resulting in a daughter DNA double helix containing one parental DNA strand and a newly synthesized strand.
Ribonucleic acid, or RNA, is mainly involved in the process of protein synthesis under the direction of DNA. RNA is usually single-stranded and is made of ribonucleotides that are linked by phosphodiester bonds. A ribonucleotide in the RNA chain contains ribose (the pentose sugar), one of the four nitrogenous bases (A, U, G, and C), and the phosphate group. Even though the RNA is single stranded, most RNA types show extensive intramolecular base pairing between complementary sequences, creating a predictable three-dimensional structure essential for their function.
There are four major types of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and microRNA (miRNA). The first, mRNA, carries the message from DNA, which controls all of the cellular activities in a cell. If a cell requires a certain protein to be synthesized, the gene for this product is turned &ldquoon&rdquo and the messenger RNA is synthesized in the nucleus. The RNA base sequence is complementary to the coding sequence of the DNA from which it has been copied. However, in RNA, the base T is absent and U is present instead. If the DNA strand has a sequence AATTGCGC, the sequence of the complementary RNA is UUAACGCG. In the cytoplasm, the mRNA interacts with ribosomes and other cellular machinery (Figure (PageIndex<3>)). More is described about their roles in later chapters.
Figure (PageIndex<3>): A ribosome has two parts: a large subunit and a small subunit. The mRNA sits in between the two subunits. A tRNA molecule recognizes a codon on the mRNA, binds to it by complementary base pairing, and adds the correct amino acid to the growing peptide chain.
Table (PageIndex<1>) below summarizes features of DNA and RNA.
Table (PageIndex<1>): Features of DNA and RNA.
|Features of DNA and RNA|
|Function||Carries genetic information||Involved in protein synthesis|
|Location||Remains in the nucleus||Leaves the nucleus|
|Structure||Double helix||Usually single-stranded|
|Pyrimidines||Cytosine, thymine||Cytosine, uracil|
|Purines||Adenine, guanine||Adenine, guanine|
As you have learned, information flow in an organism takes place from DNA to RNA to protein. DNA dictates the structure of mRNA in a process known as transcription , and RNA dictates the structure of protein in a process known as translation . This is known as the Central Dogma of Life, which holds true for all organisms however, exceptions to the rule occur in connection with viral infections.
To learn more about DNA, explore the Howard Hughes Medical Institute BioInteractive animations on the topic of DNA.
ATP is a small, relatively simple molecule (Figure (PageIndex<4>)), but within some of its bonds, it contains the potential for a quick burst of energy that can be harnessed to perform cellular work. This molecule can be thought of as the primary energy currency of cells in much the same way that money is the currency that people exchange for things they need. ATP is used to power the majority of energy-requiring cellular reactions.
Figure (PageIndex<4>): ATP is the primary energy currency of the cell. It has an adenosine backbone with three phosphate groups attached.
As its name suggests, adenosine triphosphate is comprised of adenosine bound to three phosphate groups (Figure (PageIndex<5>)). Adenosine is a nucleotide consisting of the nitrogenous base adenine and a five-carbon sugar, ribose. The three phosphate groups, in order of closest to furthest from the ribose sugar, are labeled alpha, beta, and gamma. Together, these chemical groups constitute an energy powerhouse. However, not all bonds within this molecule exist in a particularly high-energy state. Both bonds that link the phosphates are equally high-energy bonds ( phosphoanhydride bonds ) that, when broken, release sufficient energy to power a variety of cellular reactions and processes. These high-energy bonds are the bonds between the second and third (or beta and gamma) phosphate groups and between the first and second phosphate groups. The reason that these bonds are considered &ldquohigh-energy&rdquo is because the products of such bond breaking&mdashadenosine diphosphate (ADP) and one inorganic phosphate group (Pi)&mdashhave considerably lower free energy than the reactants: ATP and a water molecule. Because this reaction takes place with the use of a water molecule, it is considered a hydrolysis reaction.
Clinical Focus: Resolution
Penny stopped using her new sunscreen and applied the corticosteroid cream to her rash as directed. However, after several days, her rash had not improved and actually seemed to be getting worse. She made a follow-up appointment with her doctor, who observed a bumpy red rash and pus-filled blisters around hair follicles (Figure (PageIndex<3>)). The rash was especially concentrated in areas that would have been covered by a swimsuit. After some questioning, Penny told the physician that she had recently attended a pool party and spent some time in a hot tub. In light of this new information, the doctor suspected a case of hot tub rash, an infection frequently caused by the bacterium Pseudomonas aeruginosa, an opportunistic pathogen that can thrive in hot tubs and swimming pools, especially when the water is not sufficiently chlorinated. P. aeruginosa is the same bacterium that is associated with infections in the lungs of patients with cystic fibrosis.
The doctor collected a specimen from Penny&rsquos rash to be sent to the clinical microbiology lab. Confirmatory tests were carried out to distinguish P. aeruginosa from enteric pathogens that can also be present in pool and hot-tub water. The test included the production of the blue-green pigment pyocyanin on cetrimide agar and growth at 42 °C. Cetrimide is a selective agent that inhibits the growth of other species of microbial flora and also enhances the production of P. aeruginosa pigments pyocyanin and fluorescein, which are a characteristic blue-green and yellow-green, respectively.
Tests confirmed the presence of P. aeruginosa in Penny&rsquos skin sample, but the doctor decided not to prescribe an antibiotic. Even though P. aeruginosa is a bacterium, Pseudomonas species are generally resistant to many antibiotics. Luckily, skin infections like Penny&rsquos are usually self-limiting the rash typically lasts about 2 weeks and resolves on its own, with or without medical treatment. The doctor advised Penny to wait it out and keep using the corticosteroid cream. The cream will not kill the P. aeruginosa on Penny&rsquos skin, but it should calm her rash and minimize the itching by suppressing her body&rsquos inflammatory response to the bacteria.
Figure (PageIndex<3>): Exposure to Pseudomonas aeruginosa in the water of a pool or hot tub can sometimes cause a skin infection that manifests as &ldquohot tub rash.&rdquo (credit: modification of work by &ldquoLsupellmel&rdquo/Wikimedia Commons)
How many phosphodiester bonds are in DNA?
It is very important to know the methodology to calculate the number of phosphodiester bonds available in various DNA molecules. The number of the phosphodiester bonds present in the DNA is proportional to the number of base pairs or nucleotides present in it. Hence, after thorough research works, scientists have presented a formula to calculate the number of phosphodiester bonds (PD). The formula is as under:
Thus, in 5’-CTAGAG-3’ there are ten available phosphodiester bonds. Similarly, in 3’-GATCT-5’ the number of phosphodiester bonds present in the DNA is 8.
‘Early RNA’ reactions characterised
Researchers have provided further glimpses into the origin of life by showing how precursor nucleotide molecules could have self-polymerised without enzymes and subsequently given rise to the catalytic activity of the simplest, most ancient forms of RNA.
The idea that life on Earth descended from primitive self-replicating RNA molecules is widely accepted. But parts of the ‘RNA world’ picture have stumped scientists, including how the first nucleotides linked themselves into short chains without the catalytic activity of enzymes, and how these short sequences subsequently developed the catalytic function required to produce longer strands of RNA and eventually complex life.
Now, groups led by Judit Sponer at Masaryk University in the Czech Republic and Ernesto Di Mauro at Italy’s Institute of Molecular Biology and Pathology have proposed solutions to both problems in two separate studies. To explain the first conundrum, they fully characterised the polymerisation reaction of cyclic guanosine monophosphate (GMP) – a nucleotide present in RNA. 1
Experiments revealed that transphosphorylation drove non-enzymatic self-polymerisation of stacked cyclic GMP to lengths of 18 units in solution as well as under dehydrating conditions, which offers a plausible route for the synthesis of ancient RNA without enzymes. They suggest that a gradually desiccating environment such as a drying lagoon could induce this polymerisation.
‘We report the only known polymerisation method so far which is plausible in a prebiotic context and selectively leads to 3’,5’-linked oligomers,’ says Sponer. This linkage is important because it is the phosphodiester bond – which forms the molecular backbone of RNA – that connects each oligomer via the 3’ carbon atom of one sugar molecule with the 5’ carbon atom of another. ‘Prior to us all attempts led to mixed 2’,5’- and 3’,5’-linked oligomers, while contemporary RNA contains exclusively 3’,5’-linkages. Thus, our study also solves the problem of the origin of 3’,5’-linkage specificity of RNA,’ explains Sponer.
The team proposed a catalytic mechanism for short strands of RNA
However, their experiments achieved a yield of just 0.13% – a figure which doesn’t inspire much confidence in Jack Szostak, who studies life’s origins at Harvard University, US. ‘The prebiotic relevance of something that low yielding would be doubtful,’ he comments. However, Sponer argues that a low yield could have been enough to create the first templates required for a subsequent and more efficient template-directed polymerisation to take over.
Herein lies the second mystery that Sponer and Di Mauro’s team tackled – how it happened that the first short oligonucleotide sequences went on to develop catalytic function.
Researchers have previously reported the synthesis of catalytically active RNA molecules but they all involve long oligonucleotide chains comprising several hundred nucleotides. The problem is that such large RNA molecules must have first evolved from shorter sequences, but so far no chemical model has explained how short RNA sequences acquired the catalytic activity to do this.
‘Our experiments suggest that the onset of the catalytic activity of RNA could occur at 9-12 nucleotide long oligomers,’ says Sponer. ‘Based on these experiments we have constructed a theoretical model, which shows that imperfect base-pairing and transient formation of tetraloop-like molecular architectures enable chemical reactions that lead to simple ribozyme-like catalytic activity.’ This, he suggests, could represent the catalytic activity of the simplest, most ancient forms of RNA. 2
‘Together, these efforts help fill in a significant gap in the RNA world hypothesis,’ comments Niles Lehman from Portland State University, US. ‘They are perhaps the clearest demonstrations of these reactions to date, and they strongly support a recombinatory RNA world. The notion that RNAs polymerized spontaneously and later recombined to make catalysts is now gaining in favour.’
Di Mauro says the challenge is now to find the conditions for self-polymerisation of other cyclic nucleotides as well as mixtures, and reveals they have preliminary results for both 3’,5’ cyclic cytidine monophosphate and 3’5’ cyclic adenosine monophosphate. ‘Our two recent studies capture important parts of the story, but […] a lot of additional issues, such as replication, remain to be explored,’ he says.
Nucleic acids are molecules made up of nucleotides that direct cellular activities such as cell division and protein synthesis. Each nucleotide is made up of a pentose sugar, a nitrogenous base, and a phosphate group. There are two types of nucleic acids: DNA and RNA. DNA carries the genetic blueprint of the cell and is passed on from parents to offspring (in the form of chromosomes). It has a double-helical structure with the two strands running in opposite directions, connected by hydrogen bonds, and complementary to each other. RNA is single-stranded and is made of a pentose sugar (ribose), a nitrogenous base, and a phosphate group. RNA is involved in protein synthesis and its regulation. Messenger RNA (mRNA) is copied from the DNA, is exported from the nucleus to the cytoplasm, and contains information for the construction of proteins. Ribosomal RNA (rRNA) is a part of the ribosomes at the site of protein synthesis, whereas transfer RNA (tRNA) carries the amino acid to the site of protein synthesis. microRNA regulates the use of mRNA for protein synthesis.
Figure A mutation occurs, and cytosine is replaced with adenine. What impact do you think this will have on the DNA structure?
Figure Adenine is larger than cytosine and will not be able to base pair properly with the guanine on the opposing strand. This will cause the DNA to bulge. DNA repair enzymes may recognize the bulge and replace the incorrect nucleotide.
Chemistry of the phosphodiester bond in RNA - Biology
Cleavage of RNA phosphodiester bonds by small molecular entities : a mechanistic insight
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Endonucleolytic cleavage of the phosphodiester bonds at the ends of the transposable element and their transfer into a target DNA molecule generally requires the assembly of a synaptic complex including the Tpase, the transposon ends, and target DNA. There are two principal modes of transposition, conservative and replicative, based on whether or not the element is copied in the course of its displacement. This is dictated by the nature and order of the cleavages at the ends ( Figure 2 ): whether the transposon is liberated from its donor backbone by double strand cleavages or whether it remains attached following cleavage of only a single strand.
Figure 2 . (opposite) Transposition strategies. Transposon DNA is indicated by open boxes or shaded boxes for newly replicated transposon DNA. Donor DNA is indicated as stippled lines and target DNA as bold lines. Strand cleavage is shown as small vertical arrows. Nucleophilic attack of the phosphodiester bond by the active 3′ hydroxyl (3′OH) resulting in strand transfer is also indicated by arrows. The toothed region shown in the target DNA represents target duplications associated with insertion. DNA polarity is shown at the top of each panel. Note that in the case of IS91, the polarity of the target DNA has been inverted to facilitate drawing of the figure.
The DNA cleavage and strand joining reactions necessary for transposition of many transposable elements with Tpases of the DDE type are remarkably similar. These Tpases catalyse endonucleolytic cleavage at each 3′ transposon end to liberate 3′ OH groups, which are then used in a concerted nucleophilic attack on the target molecule. An important feature of the transposition reaction is therefore the way in which the 5′ end (second strand) is processed.
Replicative transposition entails cleavage of only one strand at each transposon end and transfer into a target site in such a way as to create a replication fork ( Figure 2 ). Some IS elements do not appear to process the second strand and simply undergo replicative transposition, or more precisely, ‘replicative integration.’ These include members of the Tn3 and IS6 families and perhaps IS1. If transposition is intermolecular, replication from the nascent fork(s) generates cointegrates (replicon fusions), where donor and target replicons are separated by a directly repeated copy of the element at each junction. Resolution of these structures to regenerate the donor and target molecules, each carrying a single copy of the element, is accomplished by recombination between the two elements. This proceeds for some transposons by site-specific recombination promoted by a specialized transposon-specific enzyme distinct from the Tpase, the ‘resolvase’ (e.g., Tn3 family), or is taken in charge by the host homologous recombination system.
In conservative or ‘cut-and-paste’ transposition, the element is excised from the donor site and reinserted into a target site without replication. This implies cleavage of both DNA strands at the ends of the element and their rejoining to target DNA to generate a simple insertion. The original donor DNA molecule is either degraded or repaired by host-specified enzymes. Different IS elements have adopted various strategies to separate themselves from the donor DNA backbone. For the IS4 family members, IS10 and IS50 ( Figure 2 ), the two breaks are not analogous. 3′ cleavage occurs before 5′ cleavage and the free 3′ OH generated by 3′ cleavage is itself used as the nucleophile in attacking the second strand. This generates a hairpin structure at the transposon ends this is subsequently hydrolyzed to regenerate the final 3′ OH ends, which will undergo transfer to the target. The free ends are retained in a relatively stable complex with Tpase and generate a noncovalently closed excised transposon circle. This mechanism is reminiscent of V(D)J recombination used in generating the immunoglobin repertoire, although the V(D)J hairpin is generated on what might be considered as the donor backbone ends. This chain of controlled consecutive reactions allows the repeated use of a single Tpase molecule bound to each end of the element.
A second strategy is used by IS2, IS3, IS150, and IS911 and presumably by other members of this large IS3 family. Here, Tpase promotes single strand cleavage at one end of the transposon and its site-specific transfer to the same strand of the opposite end ( Figure 2 ). This circularizes a single transposon strand leaving the complementary strand attached to the donor backbone. This second transposon strand is then resolved to generate a double-stranded covalently closed transposon circle, in which the transposon ends are abutted. The resolution mechanism but could involve simple cleavage and repair or replication promoted by host proteins. The covalently attached ends can then undergo simultaneous single strand cleavage and transfer to a target. This strategy of separating the transposon from its donor molecule may have also been adopted by members of the IS21 and IS30 families. While site-specific strand transfer from one end of the element to the other generates transposon circles, it can also occur between two elements carried by the same molecule. Transfer of ends between the two IS copies in a plasmid dimer, for example, would be expected to generate head-to-tail IS tandem dimers. This type of structure has been observed for IS21, IS2, IS30, and IS911, and is extremely active in transposition.
Of those ISs that do not carry a well-defined DDE triad, only IS91 has been analyzed in detail. As suggested by the similarity of its Tpase with rolling circle type replicases, IS91 appears to have adopted a polarized rolling circle transposition mode requiring a specific tetranucleotide target sequence which abuts IRR ( Figure 2 ). ‘One-ended’ transposition products occur at high frequency in the absence of IRL. They carry a constant end defined by IRR and a variable end defined by a copy of the target consensus located in the donor plasmid. It is thought that donor strand cleavage results in a covalent complex between the 5′ IRR end and Tpase and is followed by single-strand transfer into the target DNA at a site containing a consensus tetranucleotide. The attached single strand of the IS is displaced by replication in the donor molecule. Termination is triggered when the complex reaches either the 3′ IRL end or a tetranucleotide consensus sequence in the donor ( Figure 2 ). This scheme does not, however, address how the element is replicated into the target molecule.