Chlorophyll

Many important natural substances are chelates. In chelates a central metal ion is bonded to a large organic molecule, a molecule composed of carbon, hydrogen, and other elements such as oxygen and nitrogen. One such chelate is chlorophyll, the green pigment of plants. In chlorophyll the central ion is magnesium, and the large organic molecule is a porphyrin. The porphyrin contains four nitrogen atoms that form bonds to magnesium in a square planar arrangement. There are several forms of chlorophyll.

Chlorophyll is one of the most important chelates in nature. It is capable of channelling the energy of sunlight into chemical energy through the process of photosynthesis. In photosynthesis, the energy absorbed by chlorophyll transforms carbon dioxide and water into carbohydrates and oxygen.

CO₂ + H₂O (CH₂O) + O₂

In this equation, (CH₂O) is the empirical formula of carbohydrates.) The chemical energy stored by photosynthesis in carbohydrates drives biochemical reactions in nearly all living organisms.

In the photosynthetic reaction, carbon dioxide is reduced by water; in other words, electrons are transferred from water to carbon dioxide. Chlorophyll assists this transfer. When chlorophyll absorbs light energy, an electron in chlorophyll is excited from a lower energy state to a higher energy state. In this higher energy state, this electron is more readily transferred to another molecule. This starts a chain of electron-transfer steps, which ends with an electron transferred to carbon dioxide. Meanwhile, the chlorophyll which gave up an electron can accept an electron from another molecule. This is the end of a process which starts with the removal of an electron from water. Thus, chlorophyll is at the center of the photosynthetic oxidation-reduction reaction between carbon dioxide and water.

Other molecules with structures similar to that of chlorophyll play important roles in other biochemical electron-transfer (oxidation-reduction) reactions. Heme consists of a porphyrin similar to that in chlorophyll and an iron(II) ion in the center of the porphyrin. Heme is bright red. In the red blood cells of vertebrates, heme is bound to proteins forming hemoglobin. Hemoglobin combineswith oxygen in the lungs, gills, or other respiratory surfaces and releases it in the tissues. In muscle cells, myoglobin, the name given to hemoglobin in muscles, stores oxygen as an electron source for energy-releasing oxidation-reduction reactions.

Another relative of chlorophyll is vitamin B₁₂. Vitamin B₁₂ contains a cobalt ion at the center of the porphyrin. Like heme, vitamin B₁₂ is bright red. It is essential to digestion and nutritional absorption in animals. The exact way it functions is not known. Because vitamin B₁₂ is not produced by higher plants, a strictly vegetarian diet can lead to vitamin B₁₂ deficiency. However, it is produced by molds and bacteria which grow on most foods.

The intense color of chlorophyll suggests that it may be useful as a commercial pigment. In fact, chlorophyll a is a green dye (Natural Green 3) used in soaps and cosmetics. The absorption spectrum of chlorophyll (below) shows that it absorbs strongly in the red and blue-violet regions of the visible spectrum. Because it absorbs red and blue-violet light, the light it reflects and transmits appears green. Commercial pigments with structures similar to chlorophyll have been produced in a range of colors. Some of these have slightly modified porphyrins, such as having hydrogen atoms replaced with chlorine atoms. Others have different metal ions. For example, one bright blue pigment has a copper(I) ion at the center of the porphyrin and is used primarily in coloring fabrics.

Haemoglobin

Discovery

The oxygen-carrying protein hemoglobin was discovered by Otto Funke in 1851. In that year he published a series of articles in which he described growing hemoglobin crystals by successively diluting red blood cells with a solvent such as pure water, alcohol or ether, followed by slow evaporation of the solvent from the resulting protein solution.Hemoglobin’s reversible oxygenation was described a few years later by Felix Hoppe-Seyler.In 1959 Max Perutz determined the molecular structure of the molecule.This work resulted in his sharing with John Kendrew the 1962 Nobel Prize in Chemistry.

The role of Haemoglobin in the blood was discovered by physiologist Claude Bernard. The name hemoglobin is the concatenation of heme and globin, reflecting the fact that each subunit of hemoglobin is a globular protein with an embedded heme (or haem) group. Each heme group contains one iron atom, that can bind one oxygen molecule through ion-induced dipole forces. The most common type of hemoglobin in mammals contains four such subunits.

Genetics

Mutations in the genes for the hemoglobin protein in a species result in hemoglobin variants, some of which cause a group of hereditary diseases termed the hemoglobinopathies in humans. The best known is sickle-cell disease, which was the first human disease whose mechanism was understood at the molecular level. A (mostly) separate set of diseases called thalassemias involves underproduction of normal and sometimes abnormal hemoglobins, through problems and mutations in globin gene regulation. These diseases also often produce anemia.

The chemical formulas of hemoglobins vary widely across species, and even (through common mutations) slightly among subgroups of humans.

Synthesis

Hemoglobin (Hb) is synthesized in a complex series of steps. The heme part is synthesized in a series of steps in the mitochondria and the cytosol of immature red blood cells, while the globin protein parts are synthesized by ribosomes in the cytosol.Production of Hb continues in the cell throughout its early development from the proerythroblast to the reticulocyte in the bone marrow. At this point, the nucleus is lost in mammalian red blood cells, but not in birds and many other species. Even after the loss of the nucleus in mammals, residual ribosomal RNA allows further synthesis of Hb until the reticulocyte loses its RNA soon after entering the vasculature (this hemoglobin-synthetic RNA in fact gives the reticulocyte its reticulated appearance and name).

Transcription

DNA serves as the template for the synthesis of RNA much as it does for its own replication. The Steps Some 50 different protein transcription factors bind to promoter sites, usually on the 5' side of the gene to be transcribed. An enzyme, an RNA polymerase, binds to the complex of transcription factors. Working together, they open the DNA double helix. The RNA polymerase proceeds to "read" one strand moving in its 3' ? 5' direction. In eukaryotes, this requires — at least for protein-encoding genes — that the nucleosomes in front of the advancing RNA polymerase (RNAP II) be removed. A complex of proteins is responsible for this. The same complex replaces the nucleosomes after the DNA has been transcribed and RNAP II has moved on. As the RNA polymerase travels along the DNA strand, it assembles ribonucleotides (supplied as triphosphates, e.g., ATP) into a strand of RNA. Each ribonucleotide is inserted into the growing RNA strand following the rules of base pairing. Thus for each C encountered on the DNA strand, a G is inserted in the RNA; for each G, a C; and for each T, an A. However, each A on the DNA guides the insertion of the pyrimidine uracil (U, from uridine triphosphate, UTP). There is no T in RNA. Quality control. Occasionally RNA polymerase will select and insert an incorrect, mismatched, ribonucleotide. When this occurs in bacteria (and perhaps in all organisms), the enzyme backs up, removes the incorrect nucleotide (and the one preceding it) and tries again. (Described by Zenkin et al., in the 28 July 2006 issue of Science.)

Synthesis of the RNA proceeds in the 5' ? 3' direction. As each nucleoside triphosphate is brought in to add to the 3' end of the growing strand, the two terminal phosphates are removed. When transcription is complete, the transcript is released from the polymerase and, shortly thereafter, the polymerase is released from the DNA. Note that at any place in a DNA molecule, either strand may be serving as the template; that is, some genes "run" one way, some the other (and in a few remarkable cases, the same segment of double helix contains genetic information on both strands!). In all cases, however, RNA polymerase transcribes the DNA strand in its 3' ? 5' direction.

Prokaryotic vs. eukaryotic transcription

Prokaryotic transcription occurs in the cytoplasm alongside translation. Eukaryotic transcription is primarily localized to the nucleus, where it is separated from the cytoplasm by the nuclear membrane. The transcript is then transported into the cytoplasm where translation occurs. Another important difference is that eukaryotic DNA is wound around histones to form nucleosomes and packaged as chromatin. Chromatin has a strong influence on the accessibility of the DNA to transcription factors and the transcriptional machinery including RNA polymerase. In prokaryotes, mRNA is not modified. Eukaryotic mRNA is modified through RNA splicing, 5' end capping, and the addition of a polyA tail.

Reverse transcription

Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA. HIV has an RNA genome that is duplicated into DNA. The resulting DNA can be merged with the DNA genome of the host cell. The main enzyme responsible for synthesis of DNA from an RNA template is called reverse transcriptase. In the case of HIV, reverse transcriptase is responsible for synthesizing a complementary DNA strand (cDNA) to the viral RNA genome. An associated enzyme, ribonuclease H, digests the RNA strand, and reverse transcriptase synthesises a complementary strand of DNA to form a double helix DNA structure. This cDNA is integrated into the host cell's genome via another enzyme (integrase) causing the host cell to generate viral proteins which reassemble into new viral particles. Subsequently, the host cell undergoes programmed cell death (apoptosis).

Some eukaryotic cells contain an enzyme with reverse transcription activity called telomerase. Telomerase is a reverse transcriptase that lengthens the ends of linear chromosomes. Telomerase carries an RNA template from which it synthesizes DNA repeating sequence, or "junk" DNA. This repeated sequence of "junk" DNA is important because every time a linear chromosome is duplicated, it is shortened in length. With "junk" DNA at the ends of chromosomes, the shortening eliminates some repeated, or junk sequence, rather than the protein-encoding DNA sequence that is further away from the chromosome ends. Telomerase is often activated in cancer cells to enable cancer cells to duplicate their genomes without losing important protein-coding DNA sequence. Activation of telomerase can be part of the process that allows cancer cells to become immortal.

Translation

There are three setps in translation of mRNA into protine molicules, which are as follows
1. Initiation
The small subunit of the ribosome binds to a site "upstream" (on the 5' side) of the start of the message. It proceeds downstream (5' -> 3') until it encounters the start codon AUG. (The region between the cap and the AUG is known as the 5'-untranslated region [5'-UTR].) Here it is joined by the large subunit and a special initiator tRNA. The initiator tRNA binds to the P site (shown in pink) on the ribosome. In eukaryotes, initiator tRNA carries methionine (Met). (Bacteria use a modified methionine designated fMet.)
2. Elongation
An aminoacyl-tRNA (a tRNA covalently bound to its amino acid) able to base pair with the next codon on the mRNA arrives at the A site (green) associated with: an elongation factor (called EF-Tu in bacteria) GTP (the source of the needed energy)The preceding amino acid (Met at the start of translation) is covalently linked to the incoming amino acid with a peptide bond (shown in red). The initiator tRNA is released from the P site. The ribosome moves one codon downstream. This shifts the more recently-arrived tRNA, with its attached peptide, to the P site and opens the A site for the arrival of a new aminoacyl-tRNA. This last step is promoted by another protein elongation factor (called EF-G in bacteria) and the energy of another molecule of GTP. Note: the initiator tRNA is the only member of the tRNA family that can bind directly to the P site. The P site is so-named because, with the exception of initiator tRNA, it binds only to a peptidyl-tRNA molecule; that is, a tRNA with the growing peptide attached.
The A site is so-named because it binds only to the incoming aminoacyl-tRNA; that is the tRNA bringing the next amino acid. So, for example, the tRNA that brings Met into the interior of the polypeptide can bind only to the A site.
3. Termination
The end of translation occurs when the ribosome reaches one or more STOP codons (UAA, UAG, UGA). (The nucleotides from this point to the poly(A) tail make up the 3'-untranslated region [3'-UTR] of the mRNA.) There are no tRNA molecules with anticodons for STOP codons. (With a few special exceptions: link to mitochondrial genes and to nonstandard amino acids.)
However, protein release factors recognize these codons when they arrive at the A site. Binding of these proteins —along with a molecule of GTP — releases the polypeptide from the ribosome. The ribosome splits into its subunits, which can later be reassembled for another round of protein synthesis.
Regulation of Translation
The expression of most genes is controlled at the level of their transcription. Transcription factors (proteins) bind to promoters and enhancers turning on (or off) the genes they control. However, gene expression can also be controlled at the level of translation.
By General RNA-Degradation Machinery
P bodiesThe cytosol of eukaryotes contains protein complexes that compete with ribosomes for access to mRNAs. As these increase their activity, they sequester mRNAs in larger aggregates called P bodies (for "processing bodies", but this processing should not be confused with the processing of pre-mRNA to mature mRNA that occurs in the nucleus).
The repression proteins break down the mRNA by removing its "cap" removing its poly(A) tail degrading the remaining message (nibbling away in the 5' -> 3' direction) What controls the dynamic balance between ribosomes and P bodies for access to mRNAs remains to be learned. But this mechanism provides for
destruction of "bad" mRNAs (e.g., those with premature STOP codons [see Nonsense-Mediated mRNA Decay (NMD)]; turnover of mRNAs thus increasing the flexibility of gene expression in the cell.
By MicroRNAs (miRNAs)Here small RNA molecules bind to a complementary portion in the 3'-UTR of the mRNA and prevent it from being translated by ribosomes and/or trigger its destruction.
By Gene-Specific ProteinsTranslation of at least one mRNA in humans is repressed by a protein — aminoacyl tRNA synthetase. In response to the inflammatory cytokine interferon-gamma [IFN-?], the synthetase abandons its normal function (adding Glu and Pro to their respective tRNAs) and instead binds to the mRNA blocking its translation.
In some bacteria, a protein product may inhibit the further translation of its own mRNA (a kind of feedback inhibition). It does so by binding to a site which blocks the mRNA from further association with a ribosome.

Gene Splicing

Gene splicing is a post-transcriptional modification in which a single gene can code for multiple proteins. Gene Splicing is done in eukaryotes, prior to mRNA translation, by the differential inclusion or exclusion of regions of pre-mRNA. Gene splicing is an important source of protein diversity. During a typical gene splicing event, the pre-mRNA transcribed from one gene can lead to different mature mRNA molecules that generate multiple functional proteins. Thus, gene splicing enables a single gene to increase its coding capacity, allowing the synthesis of protein isoforms that are structurally and functionally distinct. Gene splicing is observed in high proportion of genes. In human cells, about 40-60% of the genes are known to exhibit alternative splicing.

Gene Splicing Mechanism: There are several types of common gene splicing events. These are the events that can simultaneously occur in the genes after the mRNA is formed from the transcription step of the central dogma of molecular biology.

Exon Skipping: This is the most common known gene splicing mechanism in which exon(s) are included or excluded from the final gene transcript leading to extended or shortened mRNA variants. The exons are the coding regions of a gene and are responsible for producing proteins that are utilized in various cell types for a number of functions.

Intron Retention: An event in which an intron is retained in the final transcript. In humans 2-5 % of the genes have been reported to retain introns. The gene splicing mechanism retains the non-coding (junk) portions of the gene and leads to a demornity in the protein structure and functionality.
Alternative 3' splice site and 5' splice site:
Alternative gene splicing includes joining of different 5' and 3' splice site. In this kind of gene splicing, two or more alternative 5' splice site compete for joining to two or more alternate 3' splice site.
Splice Variant Detection Methods
Gene splicing leads to the synthesis of alternate proteins that play an important role in the human physiology and disease. Currently, the most efficient methods for large scale detection of splice variants include computational prediction methods and microarray analysis. Microarray based splice variant detection is the most popular method currently in use. The highly parallel and sensitive nature of microarrays make them ideal for monitoring gene expression on a tissue-specific, genome-wide level. Microarray based methods for detecting splice variants provide a robust, scalable platform for high-throughput discovery of alternative gene splicing. A number of novel gene transcripts were detected using microarray based methods that were not detected by ESTs using computational methods. Another commonly used method for discovering of novel gene isoforms is RT-PCR followed by sequencing. This is a powerful approach and can be effectively used for analyzing a small number of genes. However, it only provides only a limited view of the gene structure, is labor-intensive, and does not easily scale to thousands of genes or hundreds of tissues.
Challenges in Microarray Design for Splice Variant DetectionMicroarray based gene splicing detection poses some unique challenges in designing probes for isoforms that show a high degree of homology. In order to differentiate between these isoforms, a microarray that uses a combination of probes for exons and exon-exon junctions is used. Exon skipping events or other deletions can be monitored by using junction probes. For example, a probe spanning the exon 1 and exon 3 of the gene will detect the skipping of exon 2 from the gene that is translated into a protein.

DNA replication

Before a cell can divide, it must duplicate all its DNA. In eukaryotes, this occurs during S phase of the cell cycle.
The Biochemical ReactionsDNA replication begins with the "unzipping" of the parent molecule as the hydrogen bonds between the base pairs are broken. Once exposed, the sequence of bases on each of the separated strands serves as a template to guide the insertion of a complementary set of bases on the strand being synthesized. The new strands are assembled from deoxynucleoside triphosphates. Each incoming nucleotide is covalently linked to the "free" 3' carbon atom on the pentose (figure) as the second and third phosphates are removed together as a molecule of pyrophosphate (PPi). The nucleotides are assembled in the order that complements the order of bases on the strand serving as the template. Thus each C on the template guides the insertion of a G on the new strand, each G a C, and so on. When the process is complete, two DNA molecules have been formed identical to each other and to the parent molecule.
The Enzymes
A portion of the double helix is unwound by a helicase. A molecule of a DNA polymerase binds to one strand of the DNA and begins moving along it in the 3' to 5' direction, using it as a template for assembling a leading strand of nucleotides and reforming a double helix. In eukaryotes, this molecule is called DNA polymerase delta (d). Because DNA synthesis can only occur 5' to 3', a molecule of a second type of DNA polymerase (epsilon, e, in eukaryotes) binds to the other template strand as the double helix opens. This molecule must synthesize discontinuous segments of polynucleotides (called Okazaki fragments). Another enzyme, DNA ligase I then stitches these together into the lagging strand.

DNA Replication is Semiconservative

When the replication process is complete, two DNA molecules — identical to each other and identical to the original — have been produced. Each strand of the original molecule has remained intact as it served as the template for the synthesis of a complementary strand.This mode of replication is described as semi-conservative: one-half of each new molecule of DNA is old; one-half new. Watson and Crick had suggested that this was the way the DNA would turn out to be replicated. Proof of the model came from the experiments of Meselson and Stahl.
Speed of ReplicationBacteriaThe single molecule of DNA that is the E. coli genome contains 4.7 x 106 nucleotide pairs. DNA replication begins at a single, fixed location in this molecule, the replication origin, proceeds at about 1000 nucleotides per second, and thus is done in no more than 40 minutes. And thanks to the precision of the process (which includes a "proof-reading" function), the job is done with only about one incorrect nucleotide for every 109 nucleotides inserted. In other words, more often than not, the E. coli genome (4.7 x 106) is copied without error!
Eukaryotes

The average human chromosome contains 150 x 106 nucleotide pairs which are copied at about 50 base pairs per second. The process would take a month (rather than the hour it actually does) but for the fact that there are many places on the eukaryotic chromosome where replication can begin. Replication begins at some replication origins earlier in S phase than at others, but the process is completed for all by the end of S phase. As replication nears completion, "bubbles" of newly replicated DNA meet and fuse, finally forming two new molecules.
Control of Replication

With their multiple origins, how does the eukaryotic cell know which origins have been already replicated and which still await replication?
An observation: When a cell in G2 of the cell cycle is fused with a cell in S phase, the DNA of the G2 nucleus does not begin replicating again even though replication is proceeding normally in the S-phase nucleus. Not until mitosis is completed, can freshly-synthesized DNA be replicated again.
Two control mechanisms have been identified — one positive and one negative. This redundancy probably reflects the crucial importance of precise replication to the integrity of the genome.
Licensing: positive control of replicationIn order to be replicated, each origin of replication must be bound by: an Origin Recognition Complex of proteins (ORC). These remain on the DNA throughout the process. Accessory proteins called licensing factors. These accumulate in the nucleus during G1 of the cell cycle. They include: Cdc-6 and Cdt-1, which bind to the ORC and are essential for coating the DNA with MCM proteins. Only DNA coated with MCM proteins (there are 6 of them) can be replicated. Once replication begins in S phase,
Cdc-6 and Cdt-1 leave the ORCs (the latter by ubiquination and destruction in proteasomes). The MCM proteins leave in front of the advancing replication fork. Geminin: negative control of replicationG2 nuclei also contain at least one protein — called geminin — that prevents assembly of MCM proteins on freshly-synthesized DNA (probably by blocking the actions of Cdt1).
As the cell completes mitosis, geminin is degraded so the DNA of the two daughter cells will be able to respond to licensing factors and be able to replicate their DNA at the next S phase.

DNA Structure

DNA is a polymer. The monomer units of DNA are nucleotides, and the polymer is known as a "polynucleotide." Each nucleotide consists of a 5-carbon sugar (deoxyribose), a nitrogen containing base attached to the sugar, and a phosphate group. There are four different types of nucleotides found in DNA, differing only in the nitrogenous base. The four nucleotides are given one letter abbreviations as shorthand for the four bases.
A is for adenine G is for guanine C is for cytosine T is for . Adenine and guanine are purines. Purines are the larger of the two types of bases found in DNA. Cytosine and thymine are pyrimidines. The 6 stoms (4 carbon, 2 nitrogen) are numbered 1-6. Like purines, all pyrimidine ring atoms lie in the same plane.
Deoxyribose Sugar
The deoxyribose sugar of the DNA backbone has 5 carbons and 3 oxygens. The carbon atoms are numbered 1', 2', 3', 4', and 5' to distinguish from the numbering of the atoms of the purine and pyrmidine rings. The hydroxyl groups on the 5'- and 3'- carbons link to the phosphate groups to form the DNA backbone. Deoxyribose lacks an hydroxyl group at the 2'-position when compared to ribose, the sugar component of RNA.

Nucleosides
A nucleoside is one of the four DNA bases covalently attached to the C1' position of a sugar. The sugar in deoxynucleosides is 2'-deoxyribose. The sugar in ribonucleosides is ribose. Nucleosides differ from nucleotides in that they lack phosphate groups. The four different nucleosides of DNA are deoxyadenosine (dA), deoxyguanosine (dG), deoxycytosine (dC), and (deoxy)thymidine (dT, or T).
Nucleotides
A nucleotide is a nucleoside with one or more phosphate groups covalently attached to the 3'- and/or 5'-hydroxyl group(s).
DNA Backbone
The DNA backbone is a polymer with an alternating sugar-phosphate sequence. The deoxyribose sugars are joined at both the 3'-hydroxyl and 5'-hydroxyl groups to phosphate groups in ester links, also known as "phosphodiester" bonds.
Features of the 5'-d(CGAAT) structure:
Alternating backbone of deoxyribose and phosphodiester groups Chain has a direction (known as polarity), 5'- to 3'- from top to bottom Oxygens (red atoms) of phosphates are polar and negatively charged A, G, C, and T bases can extend away from chain, and stack atop each other Bases are hydrophobic DNA Double HelixDNA is a normally double stranded macromolecule. Two polynucleotide chains, held together by weak thermodynamic forces, form a DNA molecule.