Types of RNA

Overview

Structure of a hammerhead ribozyme, a ribozyme that cuts RNA

Messenger RNA (mRNA) is the RNA that carries information from DNA to the ribosome, the sites of protein synthesis (translation) in the cell. The coding sequence of the mRNA determines the amino acid sequence in the protein that is produced.^[19] Many RNAs do not code for protein however. These non-coding RNAs can be encoded by their own genes (RNA genes), but can also derive from mRNA introns.^[20] The most prominent examples of non-coding RNAs are transfer RNA (tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of translation.^[13] There are also non-coding RNAs involved in gene regulation, RNA processing and other roles. Certain RNAs are able to catalyse chemical reactions such as cutting and ligating other RNA molecules,^[21] and the catalysis of peptide bond formation in the ribosome;^[15] these are known as ribozymes.

http://en.wikipedia.org/wiki/RNA

RNA Comparison with DNA

RNA and DNA differ in three main ways. First, unlike DNA which is double-stranded, RNA is a single-stranded molecule in most of its biological roles and has a much shorter chain of nucleotides. Second, while DNA contains deoxyribose, RNA contains ribose, (there is no hydroxyl group attached to the pentose ring in the 2' position in DNA). These hydroxyl groups make RNA less stable than DNA because it is more prone to hydrolysis. Third, the complementary nucleotide to adenine is not thymine, as it is in DNA, but rather uracil, which is an unmethylated form of thymine.^[13]

The 50S ribosomal subunit. RNA is in orange, protein in blue. The active site is in the middle (red).

Like DNA, most biologically active RNAs including tRNA, rRNA, snRNAs and other, non-coding, RNAs are extensively base paired to form double stranded helices. Structural analysis of these RNAs have revealed that they are highly structured. Unlike DNA, this structure is not long double-stranded helices but rather collections of short helices packed together into structures akin to proteins. In this fashion, RNAs can achieve chemical catalysis, like enzymes.^[14] For instance, determination of the structure of the ribosome—an enzyme that catalyzes peptide bond formation—revealed that its active site is composed entirely of RNA.

http://en.wikipedia.org/wiki/RNA

RNA Structure

Each nucleotide in RNA contains a ribose sugar, with carbons numbered 1' through 5'. A base is attached to the 1' position, generally adenine (A), cytosine (C), guanine (G) or uracil (U). Adenine and guanine are purines, cytosine and uracil are pyrimidines. A phosphate group is attached to the 3' position of one ribose and the 5' position of the next. The phosphate groups have a negative charge each at physiological pH, making RNA a charged molecule (polyanion). The bases may form hydrogen bonds between cytosine and guanine, between adenine and uracil and between guanine and uracil.^[1] However other interactions are possible, such as a group of adenine bases binding to each other in a bulge,^[2] or the GNRA tetraloop that has a guanine–adenine base-pair.^[1]

Chemical structure of RNA

An important structural feature of RNA that distinguishes it from DNA is the presence of a hydroxyl group at the 2' position of the ribose sugar. The presence of this functional group causes the helix to adopt the A-form geometry rather than the B-form most commonly observed in DNA.^[3] This results in a very deep and narrow major groove and a shallow and wide minor groove.^[4] A second consequence of the presence of the 2'-hydroxyl group is that in conformationally flexible regions of an RNA molecule (that is, not involved in formation of a double helix), it can chemically attack the adjacent phosphodiester bond to cleave the backbone.^[5]

RNA is transcribed with only four bases (adenine, cytosine, guanine and uracil),^[6] but there are numerous modified bases and sugars in mature RNAs. Pseudouridine (Ψ), in which the linkage between uracil and ribose is changed from a C–N bond to a C–C bond, and ribothymidine (T), are found in various places (most notably in the TΨC loop of tRNA).^[7] Another notable modified base is hypoxanthine, a deaminated adenine base whose nucleoside is called inosine. Inosine plays a key role in the wobble hypothesis of the genetic code.^[8] There are nearly 100 other naturally occurring modified nucleosides,^[9] of which pseudouridine and nucleosides with 2'-O-methylribose are the most common.^[10] The specific roles of many of these modifications in RNA are not fully understood. However, it is notable that in ribosomal RNA, many of the post-transcriptional modifications occur in highly functional regions, such as the peptidyl transferase center and the subunit interface, implying that they are important for normal function.^[11]

Secondary structure of a telomerase RNA

The functional form of single stranded RNA molecules, just like proteins, frequently requires a specific tertiary structure. The scaffold for this structure is provided by secondary structural elements which are hydrogen bonds within the molecule. This leads to several recognizable "domains" of secondary structure like hairpin loops, bulges and internal loops.^[12] There has been a significant amount of research directed at the RNA structure prediction problem.

Watson-Crick base pairs in a siRNA (hydrogen atoms are not shown)

http://en.wikipedia.org/wiki/RNA

Modification And Editing Of Rna

Book Description
Centre National de la recherche Scientifique. Explores ways in which cells respond to genome injury and alterations, including spontaneous and environmental. Includes comprehensive coverage of mutagenesis and human diseases and syndromes with defective cellular responses to DNA damage. Extensive references and 380 color figures. For molecular biologists.

Book Info
Centre National de la recherche Scientifique. Explores ways in which cells respond to genome injury and alterations, including spontaneous and environmental. Includes comprehensive coverage of mutagenesis and human diseases and syndromes with defective cellular responses to DNA damage. Extensive references and 380 color figures. For molecular biologists.

http://www.amazon.com/Modification-And-Editing-Of-Rna/dp/1555811337/ref=sr_1_5?ie=UTF8&s=books&qid=1207895995&sr=1-5

Fine-Tuning of RNA Functions by Modification and Editing

Naturally occurring RNA always contains numerous biochemically altered nucleotides. They are formed by enzymatic modification of the primary transcripts during the complex RNA maturation process designated RNA modification. A large number of enzymes catalyzing the formation of these modified nucleosides or converting one canonical base into another at the posttranscriptional level have been studied for many years, but only recently have systematic and comparative studies begun. The functions of individual enzymes and/or the modified/edited nucleosides in RNA, however, have remained largely ignored.

This book provides advance information on RNA modification, including the associated editing machinery, while offering the reader some perspective on the significance of such modifications in fine-tuning the structure and functions of mature RNA molecules and hence the ability to influence the efficiency and accuracy of genetic expression. Outstanding scientists who are actively working on RNA modification/editing processes have provided up-to-date information on these intriguing cellular processes that have been generated over the course of millions of years in all living organisms. Each review has been written and illustrated for a large audience of readers, not only specialists in the field, but also for advanced students or researchers who want to learn more about recent progress in RNA modification and editing.

http://www.amazon.com/Fine-Tuning-Functions-Modification-Editing-Genetics/dp/3540244956/ref=sr_1_4?ie=UTF8&s=books&qid=1207895995&sr=1-4

RNA and DNA Editing: Molecular Mechanisms and Their Integration into Biological Systems

Latest discoveries in RNA and DNA editing research, with expert forecasts of the field's future directions

RNA and DNA Editing assembles a team of leading experts who present the latestdiscoveries in the field alongside the latest models and methodology. In addition,the authors set forth the many open questions and suggest routes for further investigation. Overall, the book serves as a practical guide for professionals in the field who need to understand the interrelationship of RNA and DNA editing with other chemical and biological processes.

This book is divided into four sections, providing a clear, contextual map of the field:

Diversification of the Proteome through RNA and DNA Editing discusses the occurrence of editing sites within transcriptomes and their distribution within individual RNAs in order to explore why nucleic acid sequences are edited instead of encoded genomically

Functional Coordination of RNA Editing with Other Cellular Mechanisms brings to the forefront why RNA and DNA editing is essential for cell survival and adaptation

Predictive Studies underscores the power of computational approaches in identifying novel editing sites and predicting the biological consequences of editing at these sites

Structural Approaches presents comparative structural analyses that suggest conserved protein folds and implicate, in some instances, their ancient phylogenic origins as components of editing machinery

This book not only discusses the current state of research in depth, it also gives new contributors an opportunity to express their vision. The perspectives voiced by these authors are provocative and intended to motivate discussion and inspire new experiments. Finally, this book will promote new hypotheses and models that can serve as springboards for the next generation of discoveries in the field.

About the Author

Harold C. Smith, PhD, is Professor in the Department of Biochemistry and Biophysics at the University of Rochester and the founder and Chief Scientific Officerof OyaGen, a biotech company that develops drugs that target editing enzymes. Dr. Smith organized the first Gordon Research Conference on RNA Editing in 1997 and holds four patents.

http://www.amazon.com/RNA-DNA-Editing-Mechanisms-Integration/dp/0470109912/ref=sr_1_3?ie=UTF8&s=books&qid=1207895995&sr=1-3

DNA-RNA-Protein

DNA carries the genetic information of a cell and consists of thousands of genes. Each gene serves as a recipe on how to build a protein molecule. Proteins perform important tasks for the cell functions or serve as building blocks. The flow of information from the genes determines the protein composition and thereby the functions of the cell.

The DNA is situated in the nucleus, organized into chromosomes. Every cell must contain the genetic information and the DNA is therefore duplicated before a cell divides (replication). When proteins are needed, the corresponding genes are transcribed into RNA (transcription). The RNA is first processed so that non-coding parts are removed (processing) and is then transported out of the nucleus (transport). Outside the nucleus, the proteins are built based upon the code in the RNA (translation).

http://nobelprize.org/educational_games/medicine/dna/index.html

RNA

Ribonucleic acid or RNA is a nucleic acid made from a long chain of nucleotide units. Each nucleotide consists of a nitrogenous base, a ribose sugar, and a phosphate. RNA is very similar to DNA, but differs in a few important structural details: in the cell RNA is usually single stranded, while DNA is usually double stranded. RNA nucleotides contain ribose while DNA contains deoxyribose (a type of ribose that lacks one oxygen atom), and in RNA the nucleotide uracil substitutes for thymine, which is present in DNA.

RNA is transcribed from DNA by enzymes called RNA polymerases and is generally further processed by other enzymes. Some of these RNA-processing enzymes contain RNA as part of their structures. RNA is also central to the translation of some RNAs into proteins. In this process, a type of RNA called messenger RNA carries information from DNA to structures called ribosomes. These ribosomes are made from proteins and ribosomal RNAs, which come together to form a molecular machine that can read messenger RNAs and translate the information they carry into proteins. It has also been known since the 1990s that several types of RNA regulate which genes are active.

A hairpin loop from a pre-mRNA. Notice its nitrogen-rich (blue) bases and oxygen-rich (red) backbone.

http://en.wikipedia.org/wiki/RNA

STRUCTURE AND FUNCTION OF RNA

RNA is structurally similar to DNA!

Both nucleic acids are sugar-phosphate polymers and both have nitrogen bases attached to the sugars of the backbone- but there are several important differences.

• They differ in composition:

1. The sugar in RNA is ribose, not the deoxyribose in DNA (as we previously learned).
2. The base uracil is present in RNA instead of thymine.
   

• They also differ in size and structure:

1. RNA molecules are smaller (shorter) than DNA molecules,
2. RNA is single-stranded, not double-stranded like DNA.
   

• Another difference between RNA and DNA is in function. DNA has only one function-STORING GENETIC INFORMATION in its sequence of nucleotide bases. But there are three main kinds of ribonucleic acid, each of which has a specific job to do.

1. Ribosomal RNAs-exist outside the nucleus in the cytoplasm of a cell in structures called ribosomes. Ribosomes are small, granular structures where protein synthesis takes place. Each ribosome is a complex consisting of about 60% ribosomal RNA (rRNA) and 40% protein.
2. Messenger RNAs-are the nucleic acids that "record" information from DNA in the cell nucleus and carry it to the ribosomes and are known as messenger RNAs (mRNA).
3. Transfer RNAs-The function of transfer RNAs (tRNA) is to deliver amino acids one by one to protein chains growing at ribosomes.

http://ncc.gmu.edu/dna/rna.htm

What is the difference between DNA and RNA?

DNA and RNA Introduction

Introduction:

The nucleic acids are informational molecules because their primary structure contains a code or set of directions by which they can duplicate themselves and guide the synthesis of proteins. The synthesis of proteins - most of which are enzymes - ultimately governs the metabolic activities of the cell. In 1953, Watson, an American biologist, and Crick, an English biologist, proposed the double helix structure for DNA. This development set the stage for a new and continuing era of chemical and biological investigation. The two main events in the life of a cell - dividing to make exact copies of themselves, and manufacturing proteins - both rely on blueprints coded in our genes.

There are two types of nucleic acids which are polymers found in all living cells. Deoxyribonucleic Acid (DNA) is found mainly in the nucleus of the cell, while Ribonucleic Acid (RNA) is found mainly in the cytoplasm of the cell although it is usually synthesized in the nucleus. DNA contains the genetic codes to make RNA and the RNA in turn then contains the codes for the primary sequence of amino acids to make proteins.

Nucleic Acid Parts List:

The best way to understand the structures of DNA and RNA is to identify and examine individual parts of the structures first. The complete hydrolysis of nucleic acids yields three major classes of compounds: pentose sugars, phosphates, and heterocyclic amines (or bases).

Phosphate: A major requirement of all living things is a suitable source of phosphorus. One of the major uses for phosphorus is as the phosphate ion which is incorporated into DNA and RNA.

Pentose Sugars:

There are two types of pentose sugars found in nucleic acids. This difference is reflected in their names--deoxyribonucleic acid indicates the presence of deoxyribose; while ribonucleic acid indicates the presence of ribose.

In the graphic on the left, the structures of both ribose and deoxyribose are shown. Note the red -OH on one and the red -H on the other are the only differences. The alpha and beta designations are interchangeable and are not a significant difference between the two.

Heterocyclic Amines:

Heterocyclic amines are sometimes called nitrogen bases or simply bases. The heterocyclic amines are derived from two root structures: purines or pyrimidines. The purine root has both a six and a five member ring; the pyrimidine has a single six member ring.

There are two major purines, adenine (A) and guanine (G), and three major pyrimidines, cytosine (C), uracil (U), and thymine (T). The structures are shown in the graphic on the left. As you can see, these structures are called "bases" because the amine groups as part of the ring or as a side chain have a basic property in water.

A major difference between DNA and RNA is that DNA contains thymine, but not uracil, while RNA contains uracil but not thymine. The other three heterocyclic amines, adenine, guanine, and cytosine are found in both DNA and RNA. For convenience, you may remember, the list of heterocyclic amines in DNA by the words: The Amazing Gene Code (TAGC).

http://www.elmhurst.edu/~chm/vchembook/580DNA.html

DNA vs. RNA

What's the difference between DNA and RNA?

Both DNA and RNA are composed of repeating units of nucleotides. Each
nucleotide consists of a sugar, a phosphate and a nucleic acid base.
The sugar in DNA is deoxyribose. The sugar in RNA is ribose, the same
as deoxyribose but with one more OH (oxygen-hydrogen atom combination
called a hydroxyl). This is the biggest difference between DNA and RNA.
Another difference is that RNA molecules can have a much greater variety
of nucleic acid bases. DNA has mostly just 4 different bases with a few
extra occasionally.  The difference in these bases (between DNA and RNA)
allows RNA molecules to assume a wide variety of shapes and also  many
different functions. DNA, on the other hand, serves as a set of directions
and that's about all (but that's absolutely necessary!). ---DrPam

http://www.newton.dep.anl.gov/askasci/bio99/bio99410.htm

Does the fountain of youth spring from our chromosomes?

The search for immortality has long been a quest of the human spirit. Whether it manifests as a belief in some sort of spiritual afterlife or in prolonging our mortal lives, humanity seems to find the ending of consciousness a horrid thought. Naturally, the mechanisms for why people grow old and die would gain a huge amount of attention by both researchers and the non-scientific community. Many scientists believe the key to mortality has already been found, and it is located at the ends of our chromosomes. Research has discovered that regions of repetitive DNA stretches called telomeres found on the ends of our DNA strands are cut shorter every time they are copied. Eventually the telomeres are worn away and genes near the end of the chromosomes are lost which contain protein instructions the body desperately needs to survive. Some believe that it is this slow yet eventual erosion of the ends of our chromosomes that leads to aging. The discoveries in this area will have a huge impact on more than just showing the way to a possible fountain of youth. Cancer research and cloning may also hinge on developments in the field of telomere research.

Telomeres exist as the body’s way of solving a problem with DNA replication. DNA is replicated by the use of an enzyme called DNA polymerase. DNA polymerase functions to copy our chromosomal DNA, using an existing DNA "parental" strand as a template. The polymerase performs this feat by attaching nucleotides to polymerize a new "daughter" DNA strand in complement to the parental strand. Adenine (A) is added to the new strand complementary to thymine (T) while guanine (G) is added complementary to cytosine (C), and visa versa. There are two main problems with the capabilities of the DNA polymerase. The first is that it can’t start from scratch. There must be a segment of the new strand from which the polymerase can begin attaching new nucleotides. The use of primers easily solves this problem. These primers are RNA fragments that bind by random assortment complementary to sites on the parent strand of DNA, and must be in place before the DNA polymerase can begin copying the parent strand.

The second problem caused by DNA polymerase during replication is much more difficult for cells to surpass. DNA polymerase can only work in a 5’ to 3’ direction. The terms 5’ and 3’ refer to the sugar molecule in the sugar/phosphate DNA backbone. The numbers relate to the carbon in that ring of sugar. 5’ is the fifth carbon in that ring. 3’ is the third. In order for the polymerase to attach a new complementary nucleotide, an alcohol (-OH) group must be available on the 3’ carbon of the sugar molecule. That is the site where the polymerase attaches the phosphate group of the next nucleotide. This phosphate group is subsequently attached to the 5’ carbon of the new nucleotide’s sugar group. The polymerase can therefore only work from the direction of the previously attached 5’ carbon to the 3’ carbon, which has the –OH group available for the attachment of the next nucleotide. The problem with this unidirectional movement lies with the primers, for they can’t stay in the new strand because they are RNA, and not DNA. Removal of these RNA primers is really not a problem when they are located in the middle of the new daughter strand. There will be a 5’ carbon available for a DNA polymerase to fill in the gap that remained after primer removal. However, the problem lies at the beginning each chromosome. A primer was necessary to provide a 5’ carbon for the beginning of synthesis, yet once it is removed, an upstream 5’ carbon is not available from which a polymerase can attach nucleotides and fill in the gap. Therefore, because the nucleotides are are not replaced after removal of the first primer at the beginning of every chromosome, every time the chromosome replicates the daughter strand will be shorter than the parental strand. Studies have shown that the length of a chromosome shortens by about 50 nucleotides every time it replicates. The damage isn’t huge compared to the overall length of a chromosome, but it does mean the chromosome is mortal in that it is slowly being eaten away at the ends with every cell division. If any of these 50 nucleotides contains the instruction to begin the transcription of a gene, that gene and the protein it encodes will never be usable by the body again.

Replication shortens the chromosome

The body’s natural cure to this dilemma is the production of expendable nucleotides at the 3’ end of every chromosome. These "cannon fodder" nucleotides are called telomeres. Telomeres are repetitive hexameric (6 base pair) sequences of DNA. In humans this repeated G-rich sequence is AGGGTT. These sequences are 1000-1700 base pairs long at the beginning of a mammalian life. Cells seldom survive past about 50 divisions in vitro, which most researchers ascribe to the deletion of too many genes in the process of replication.

Oddly, these telomeres are not encoded in the initial DNA resulting from egg fertilization. What this means is that the telomeres must be added later in development. In 1985 Elizabeth Blackburn and Carol Greider discovered a new DNA polymerase which can add telomeres to DNA. This polymerase, called telomerase, is a ribonucleoprotein present in the very early stages of development. Telomerase activity stops in later development, as it is only required to put the telomeres in place once. Ribonucleoproteins contain RNA, which telomerase uses as a template to synthesize the hexameric DNA telomeres. Because telomerase is a polymerase that copies an RNA template (its own) into DNA, it is a reverse transcriptase. A reverse transcriptase is so named because it is capable of writing codes of DNA from an RNA template which is the reverse of transcription. Reverse transcriptases have gained a lot of fame because they are used by retroviruses, notably HIV, for viral replication.

Telomerase binds to the 3’ end of a chromosome and lines its own RNA template so that a few of its RNA base pairs are complementary to that of the strand. Another segment of the ribozyme hangs over the edge providing a template for the synthesis of the telomeres (CCUAAC). Telomerase synthesizes the hexomeric sequence and then translocates to a new 3’ recognition site, which is within the hexanucleotide it just produced, and repeats the procedure. A normal DNA polymerase and primer can then complete the complementary strand’s 5’ end with all of the new hexomeric repeats--all except the last bit of course. The exact details of telomerase function are currently under research, but its currently understood mechanism as a DNA polymerase that carries its own template appears quite unique and phenomenal.

Could the "Fountain of Youth," simply be a shot of telomerase? Some research hints that this might be a good start to combat aging. For example, recent studies have shown that mice deficient in the gene for encoding telomerase RNA (mTR) developed liver cirrhosis sooner and regenerated much slower than normal mice. These same mice also showed improved liver function upon receiving gene delivery of telomerase. In the future, it may be possible to induce telomerase to reset aging cells back to their chromosomal state during a person’s young and vibrant 20’s. However, in most cases the addition of telomerase into somatic cells late in development would be a death sentence. Cell death (apoptosis) is often a good thing in the body. If some cells didn’t die, some tissues would never stop growing. Apoptosis is a crucial tool used by the body to maintain proper development. Certain cells must die at certain times or else the entire organism will perish.

Another aging-related subject that telomere research might prove helpful to is cloning research. Cloning researchers have found that unfortunately the telomeres of cloned animals (such as the famed cloned sheep named "Dolly") are much shorter than a counterpart of the same developmental "age". Even though cloning technology has attained successful birth rates as high as 80%, most of these clones die before even reaching adulthood. Shortened telomeres appear to be the most likely cause of these deaths. Research seeks to uncover a means of safely extending the telomeres of the clones. Some may hope that the solution to the clone problem will eventually bring about a magic youth potion to humanity.

Telomerases might be cancer's Achilles heel

Besides the prevention of age-related health problems, another motivating drive for telomerase research is to develop effective cancer treatments. Scientists are attempting to destroy the telomeres by eradicating telomerase activity in cancer cells. The purpose is to limit the number of divisions possible in these cells. Normal somatic cells have no telomerase present in them because the expression of the telomerase gene is shut down early in life. Because these cells live a long time in the body, the telomeres created early in life are long enough to serve them for the number of divisions they need to make during the lifetime of the organism. However, cancer cells are defined by unbridled cell division, and therefore it is the telomerase which allows cancer cells to continue their unhindered proliferation and subsequent immortality. One of the mutations that leads to a cell becoming cancerous is one that disrupts the cells ability to shut down telomerase expression. Cancer researchers have become very interested in designing drugs that target and inactivate telomerase, for if telomerase could be inactivated this would lead to cancer cells becoming mortal again and stop them in their tracks.

Although using telomere research for finding a treatment for cancer is a popular concept that everyone supports, the idea of significantly extending life is much more controversial. With the population of Earth bulging proudly over 6 billion souls one has to ponder if human immortality would be a blessing at this point in time. Endless life could be to society what cell immortality is to the body.

http://www3.interscience.wiley.com:8100/legacy/college/boyer/0471661791/cutting_edge/telomeres/telomeres.htm

Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein.

microRNAs (miRNAs) are single-stranded, 21- to 23-nucleotide cellular RNAs that control the expression of cognate target genes. Primary miRNA (pri-miRNA) transcripts are transformed to mature miRNA by the successive actions of two RNase III endonucleases. Drosha converts pri-miRNA transcripts to precursor miRNA (pre-miRNA); Dicer, in turn, converts pre-miRNA to mature miRNA. Here, we show that normal processing of Drosophila pre-miRNAs by Dicer-1 requires the double-stranded RNA-binding domain (dsRBD) protein Loquacious (Loqs), a homolog of human TRBP, a protein first identified as binding the HIV trans-activator RNA (TAR). Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqs(f00791) mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis.

http://www.doaj.org/doaj?func=abstract&id=121871

History of DNA research

Further information: History of molecular biology

DNA was first isolated by the Swiss physician Friedrich Miescher who, in 1869, discovered a microscopic substance in the pus of discarded surgical bandages. As it resided in the nuclei of cells, he called it "nuclein".^[117] In 1919 this discovery was followed by Phoebus Levene's identification of the base, sugar and phosphate nucleotide unit.^[118] Levene suggested that DNA consisted of a string of nucleotide units linked together through the phosphate groups. However, Levene thought the chain was short and the bases repeated in a fixed order. In 1937 William Astbury produced the first X-ray diffraction patterns that showed that DNA had a regular structure.^[119]

In 1928, Frederick Griffith discovered that traits of the "smooth" form of the Pneumococcus could be transferred to the "rough" form of the same bacteria by mixing killed "smooth" bacteria with the live "rough" form.^[120] This system provided the first clear suggestion that DNA carried genetic information, when Oswald Avery, along with coworkers Colin MacLeod and Maclyn McCarty, identified DNA as the transforming principle in 1943.^[121] DNA's role in heredity was confirmed in 1952, when Alfred Hershey and Martha Chase in the Hershey-Chase experiment showed that DNA is the genetic material of the T2 phage.^[122]

In 1953, based on X-ray diffraction images^[123] taken by Rosalind Franklin and the information that the bases were paired, James D. Watson and Francis Crick suggested^[123] what is now accepted as the first accurate model of DNA structure in the journal Nature.^[5] Experimental evidence for Watson and Crick's model were published in a series of five articles in the same issue of Nature.^[124] Of these, Franklin and Raymond Gosling's paper was the first publication of X-ray diffraction data that supported the Watson and Crick model,^[125][126] this issue also contained an article on DNA structure by Maurice Wilkins and his colleagues.^[127] In 1962, after Franklin's death, Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine.^[128] However, debate continues on who should receive credit for the discovery, as the Watson and Crick article in Nature was based on Franklin's data without either acknowledgment or her knowledge.^[129]

In an influential presentation in 1957, Crick laid out the "Central Dogma" of molecular biology, which foretold the relationship between DNA, RNA, and proteins, and articulated the "adaptor hypothesis".^[130] Final confirmation of the replication mechanism that was implied by the double-helical structure followed in 1958 through the Meselson-Stahl experiment.^[131] Further work by Crick and coworkers showed that the genetic code was based on non-overlapping triplets of bases, called codons, allowing Har Gobind Khorana, Robert W. Holley and Marshall Warren Nirenberg to decipher the genetic code.^[132] These findings represent the birth of molecular biology.

http://en.wikipedia.org/wiki/DNA

Evolution of DNA metabolism

Further information: RNA world hypothesis

DNA contains the genetic information that allows all modern living things to function, grow and reproduce. However, it is unclear how long in the 4-billion-year history of life DNA has performed this function, as it has been proposed that the earliest forms of life may have used RNA as their genetic material.^[81][93] RNA may have acted as the central part of early cell metabolism as it can both transmit genetic information and carry out catalysis as part of ribozymes.^[94] This ancient RNA world where nucleic acid would have been used for both catalysis and genetics may have influenced the evolution of the current genetic code based on four nucleotide bases. This would occur since the number of unique bases in such an organism is a trade-off between a small number of bases increasing replication accuracy and a large number of bases increasing the catalytic efficiency of ribosomes.^[95]

Unfortunately, there is no direct evidence of ancient genetic systems, as recovery of DNA from most fossils is impossible. This is because DNA will survive in the environment for less than one million years and slowly degrades into short fragments in solution.^[96] Claims for older DNA have been made, most notably a report of the isolation of a viable bacterium from a salt crystal 250-million years old,^[97] but these claims are controversial.^[98][99]

http://en.wikipedia.org/wiki/DNA

DNA-binding proteins

DNA-binding proteins

Interaction of DNA with histones (shown in white, top). These proteins' basic amino acids (below left, blue) bind to the acidic phosphate groups on DNA (below right, red).

Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called chromatin. In eukaryotes this structure involves DNA binding to a complex of small basic proteins called histones, while in prokaryotes multiple types of proteins are involved.^[63][64] The histones form a disk-shaped complex called a nucleosome, which contains two complete turns of double-stranded DNA wrapped around its surface. These non-specific interactions are formed through basic residues in the histones making ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are therefore largely independent of the base sequence.^[65] Chemical modifications of these basic amino acid residues include methylation, phosphorylation and acetylation.^[66] These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA more or less accessible to transcription factors and changing the rate of transcription.^[67] Other non-specific DNA-binding proteins found in chromatin include the high-mobility group proteins, which bind preferentially to bent or distorted DNA.^[68] These proteins are important in bending arrays of nucleosomes and arranging them into more complex chromatin structures.^[69]

A distinct group of DNA-binding proteins are the single-stranded-DNA-binding proteins that specifically bind single-stranded DNA. In humans, replication protein A is the best-characterised member of this family and is essential for most processes where the double helix is separated, including DNA replication, recombination and DNA repair.^[70] These binding proteins seem to stabilize single-stranded DNA and protect it from forming stem-loops or being degraded by nucleases.

The lambda repressor helix-turn-helix transcription factor bound to its DNA target^[71]

In contrast, other proteins have evolved to specifically bind particular DNA sequences. The most intensively studied of these are the various classes of transcription factors, which are proteins that regulate transcription. Each one of these proteins bind to one particular set of DNA sequences and thereby activates or inhibits the transcription of genes with these sequences close to their promoters. The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase responsible for transcription, either directly or through other mediator proteins; this locates the polymerase at the promoter and allows it to begin transcription.^[72] Alternatively, transcription factors can bind enzymes that modify the histones at the promoter; this will change the accessibility of the DNA template to the polymerase.^[73]

As these DNA targets can occur throughout an organism's genome, changes in the activity of one type of transcription factor can affect thousands of genes.^[74] Consequently, these proteins are often the targets of the signal transduction processes that mediate responses to environmental changes or cellular differentiation and development. The specificity of these transcription factors' interactions with DNA come from the proteins making multiple contacts to the edges of the DNA bases, allowing them to "read" the DNA sequence. Most of these base-interactions are made in the major groove, where the bases are most accessible.^[75]

The restriction enzyme EcoRV (green) in a complex with its substrate DNA^[76]

DNA-modifying enzymes

Nucleases and ligases

Nucleases are enzymes that cut DNA strands by catalyzing the hydrolysis of the phosphodiester bonds. Nucleases that hydrolyse nucleotides from the ends of DNA strands are called exonucleases, while endonucleases cut within strands. The most frequently-used nucleases in molecular biology are the restriction endonucleases, which cut DNA at specific sequences. For instance, the EcoRV enzyme shown to the left recognizes the 6-base sequence 5′-GAT|ATC-3′ and makes a cut at the vertical line. In nature, these enzymes protect bacteria against phage infection by digesting the phage DNA when it enters the bacterial cell, acting as part of the restriction modification system.^[77] In technology, these sequence-specific nucleases are used in molecular cloning and DNA fingerprinting.

Enzymes called DNA ligases can rejoin cut or broken DNA strands.^[78] Ligases are particularly important in lagging strand DNA replication, as they join together the short segments of DNA produced at the replication fork into a complete copy of the DNA template. They are also used in DNA repair and genetic recombination.^[78]

Topoisomerases and helicases

Topoisomerases are enzymes with both nuclease and ligase activity. These proteins change the amount of supercoiling in DNA. Some of these enzyme work by cutting the DNA helix and allowing one section to rotate, thereby reducing its level of supercoiling; the enzyme then seals the DNA break.^[23] Other types of these enzymes are capable of cutting one DNA helix and then passing a second strand of DNA through this break, before rejoining the helix.^[79] Topoisomerases are required for many processes involving DNA, such as DNA replication and transcription.^[24]

Helicases are proteins that are a type of molecular motor. They use the chemical energy in nucleoside triphosphates, predominantly ATP, to break hydrogen bonds between bases and unwind the DNA double helix into single strands.^[80] These enzymes are essential for most processes where enzymes need to access the DNA bases.

Polymerases

Polymerases are enzymes that synthesize polynucleotide chains from nucleoside triphosphates. The sequence of their products are copies of existing polynucleotide chains - which are called templates. These enzymes function by adding nucleotides onto the 3′ hydroxyl group of the previous nucleotide in a DNA strand. Consequently, all polymerases work in a 5′ to 3′ direction.^[81] In the active site of these enzymes, the incoming nucleoside triphosphate base-pairs to the template: this allows polymerases to accurately synthesize the complementary strand of their template. Polymerases are classified according to the type of template that they use.

In DNA replication, a DNA-dependent DNA polymerase makes a DNA copy of a DNA sequence. Accuracy is vital in this process, so many of these polymerases have a proofreading activity. Here, the polymerase recognizes the occasional mistakes in the synthesis reaction by the lack of base pairing between the mismatched nucleotides. If a mismatch is detected, a 3′ to 5′ exonuclease activity is activated and the incorrect base removed.^[82] In most organisms DNA polymerases function in a large complex called the replisome that contains multiple accessory subunits, such as the DNA clamp or helicases.^[83]

RNA-dependent DNA polymerases are a specialized class of polymerases that copy the sequence of an RNA strand into DNA. They include reverse transcriptase, which is a viral enzyme involved in the infection of cells by retroviruses, and telomerase, which is required for the replication of telomeres.^[84][32] Telomerase is an unusual polymerase because it contains its own RNA template as part of its structure.^[33]

Transcription is carried out by a DNA-dependent RNA polymerase that copies the sequence of a DNA strand into RNA. To begin transcribing a gene, the RNA polymerase binds to a sequence of DNA called a promoter and separates the DNA strands. It then copies the gene sequence into a messenger RNA transcript until it reaches a region of DNA called the terminator, where it halts and detaches from the DNA. As with human DNA-dependent DNA polymerases, RNA polymerase II, the enzyme that transcribes most of the genes in the human genome, operates as part of a large protein complex with multiple regulatory and accessory subunits.^[85]

http://en.wikipedia.org/wiki/DNA

DNA

Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information. DNA is often compared to a set of blueprints, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information.

Chemically, DNA is a long polymer of simple units called nucleotides, with a backbone made of sugars and phosphate groups joined by ester bonds. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription.

Within cells, DNA is organized into structures called chromosomes. These chromosomes are duplicated before cells divide, in a process called DNA replication. Eukaryotic organisms (animals, plants, and fungi) store their DNA inside the cell nucleus, while in prokaryotes (bacteria and archae) it is found in the cell's cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.

The structure of part of a DNA double helix


http://en.wikipedia.org/wiki/DNA

Molecular Biology Current Innovations and Future Trends

One could be led to believe that a molecular biologist armed with a copy of 'Maniatis', or one of the 'Current Protocols' publications, would have adequate technical support to successfully accomplish most experimental procedures. In the real laboratory world, we know that even established methodology is adapting and changing at an alarming rate and that new experimental approaches are regularly appearing on the horizon. This small book fills an important niche in the market, for it aims, and I believe succeeds, in bringing the reader up to date with recent innovations in established techniques as well as introducing us to more state of the art methodology.

The book contains ten chapters, all written by experts in the particular fields and interestingly, the editors have recruited over half the authors from the commercial sector. These contributions tend to bias their chapters towards products available from their particular companies, although in general they seem to have covered their subjects fairly comprehensively. Each chapter covers a review of the technique, concentrating on recent innovations and then discusses likely future trends. Most chapters end with protocols covering recent advances or more specialised approaches. Each chapter is also accompanied by an extensive list of references, in most cases concentrating on papers published in the last five years. All chapters refer to material published last year, which is a good indication that the editors and the publisher have succeeded in bringing this book to the bookshelves without undue delay.

The first chapter covers general PCR techniques and is written by a group of authors from Stratagene. In addition to covering recent advances in PCR methodology and instrumentation, the authors describe specific techniques such as cloning PCR-generated fragments and using PCR for site-directed mutagenesis. Sadly the accompanying figures are black and white copies of coloured diagrams from the company¹s catalogue and some of the detail has been lost during reproduction. A specific utilisation of PCR, thermal cycle sequencing, is described in the next chapter, which contains a generalised protocol for the technique. This is followed by a chapter devoted to methods for isolating plasmid DNA from mini-preps using silica-based resins. Whilst there are a profusion of commercial kits available, the author very rightly draws attention to the dangers of total reliance on these products and so presents a very extensive protocol utilising common laboratory reagents and equipment.

Electrophoresis is covered by three chapters, the first by Branko Kozulic, who provides a very readable account of recent theories which attempt to explain electrophoretic phenomena, including his own Œdoor-corridor¹ model. He also provides a tantalizing glimpse into the world of new gel matrices and intercalating dyes. The second chapter is devoted to pulsed field gel electrophoresis (PFGE) in which the authors review the various aspects of the technique and provide protocols for the preparation of high molecular weight DNA from soya bean leaves and provide physical mapping data from PFGE combined with two dimensional electrophoresis. The other chapter describes capillary electrophoresis (CE) as applied to the isoelectric focusing of proteins and provides an extensive protocol and a troubleshooting chart.

A chapter on subtractive hybridisation describes the use of commercially available multipurpose cloning vectors to perform cDNA subtractive hybridisation between biotinylated RNA and single stranded DNA. The unhybridised product is purified by streptavidin and used for transformation. This technique should appeal to researchers involved in gene expression and developmental studies.

The widespread use of PCR in molecular biology has required the simultaneous development of reliable methods for the production of oligo primers. A chapter describes recent developments in the related field of oligoribonucleotide synthesis. The demand for synthesized RNA is likely to increase as interest in antisense RNA and the possible use of ribozymes in gene therapy intensifies.

Finally, there are two interesting chapters on instrumentation. One describes state of the art devices for automated DNA hybridization and detection and the other is devoted to a relatively new technique called matrix assisted laser desorption ionization mass spectrometry (MALDI). The authors speculate that MALDI will, in the not too distant future, replace gel electrophoresis in the analysis of DNA sequencing reactions.

This modestly priced book provides the molecular biologist with a wealth of current information on a wide variety of essential techniques. I look forward to the publication of volume 2 in this series, later this year.

http://www.horizonpress.com/hsp/revs/revs1mb.html