Molecular biologyField of science concerned with thechemical structures and processes ofbiological phenomena at the molecularlevel. Having developed out of therelated fields of biochemistry,genetics, and biophysics, thediscipline is particularly concernedwith the study of proteins, nucleicacids, and enzymes. In the early1950s, growing knowledge of thestructure of proteins enabled thestructure of DNA to be described.
The discovery in the 1970s of certaintypes of enzymes that can cut andrecombine segments of DNA(recombination) in the chromosomes ofcertain bacteria made recombinant-DNAtechnology possible. Molecularbiologists use that technology to isolateand modify specific gene.
• Nucleic acids and nucleoprotein structure.• Replication.• Transcription.• Regulation of gene expression.• Restriction enzymes & its function in DNA technology.• Gene cloning .• Production of recombinant plasmid.• Construction of genomic and DNA libraries.• Analyzing & sequencing cloned DNA.• Analysis of specific nucleic acids in complex mixtures• polymerase chain reaction (PCR),mutation .
• In living organisms, DNA does not usually exist as a single molecule, but instead as a tightly-associated pair of molecules. These two long strands entwine like vines, in the shape of a double helix. The nucleotide repeats contain both the segment of the backbone of the molecule, which holds the chain together, and a base, which interacts with the other DNA strand in the helix.
In general, a base linked to a sugar is called anucleoside and a base linked to a sugar and oneor more phosphate groups is called anucleotide. If multiple nucleotides are linkedtogether, as in DNA, this polymer is referred toas a polynucleotide. Nucleic acids are polymericmacromolecules made from nucleotide monomers. InDNA, the purine bases are adenine and guanine, whilethe pyrimidines are thymine and cytosine. RNA usesuracil in place of thymine.
• Nucleotide structure• A nucleotide is composed of a nucleobase (nitrogenous base), a five-carbon sugar (either ribose or 2-deoxyribose), and one to three phosphate groups. Together, the nucleobase and sugar comprise a nucleoside. The phosphate groups form bonds with either the 2, 3, or 5-carbon of the sugar, with the 5-carbon site most common. Cyclic nucleotides form when the phosphate group is bound to two of the sugars hydroxyl groups. Ribonucleotides are nucleotides where the sugar is ribose, and deoxyribonucleotides contain the sugar deoxyribose. Nucleotides can contain either a purine or pyrimidine base.
SynthesisNucleotides can be synthesized by a variety of meansboth in vitro and in vivo. In vivo, nucleotides can besynthesised de novo or recycled through salvagepathways. Nucleotides undergo breakdown such thatuseful parts can be reused in synthesis reactions tocreate new nucleotides. In vitro, protecting groups maybe used during laboratory production of nucleotides. Apurified nucleoside is protected to create aphosphoramidite, which can then be used to obtainanalogues not found in nature and/or to synthesize anoligonucleotide
DNAs duplex nature• DNA is normally double-stranded. The sequences of the two strands are related so that an A on one strand is matched by a T on the other strand; likewise, a G on one strand is matched by a C on the other strand. Thus, the fraction of bases in an organisms DNA that are A is equal to the fraction of bases that are T, and the fraction of bases that are G is equal to the fraction of bases that are C. For example, if one-third of the bases are A, one-third must be T, and because the amount of G equals the amount of C, one-sixth of the bases will be G and one-sixth will be C. The importance of this relationship, termed Chargraffs rules, was recognized by Watson and Crick, who proposed that the two strands form a double helix with the two strands arranged in an antiparallel fashion, interwound head-to-tail
• In a double helix the direction of the nucleotides in one strand is opposite to their direction in the other strand. This arrangement of DNA strands is called antiparallel. The asymmetric ends of DNA strands are referred to as the 5′ (five prime) and 3′ (three prime) ends.• One of the major differences between DNA and RNA is the sugar, with 2-deoxyribose being replaced by the alternative pentose sugar ribose in RNA.• Usually,we read nucleic acid sequences of DNA in a 5′ to 3′ direction, so a DNA dinucleotide of (51) adenosine-guanosine (31) is read as AG.• The complementary sequence is CT, because both sequences are read in the 5′ to 3′ direction. The terms 5′ and 3′ refer to the numbers of the carbons on the sugar portion of the nucleotide (the base is attached to the 1′ carbon of the sugar).
• 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.
Pyrimidine ribonucleotides Pyrimidine nucleotide synthesis starts with the formation of carbamoyl phosphate from glutamine and CO2. The cyclisation reaction between carbamoyl phosphate reacts with aspartate yielding orotate in subsequent steps. Orotate reacts with 5-phosphoribosyl α-diphosphate (PRPP) yielding orotidine monophosphate (OMP) which is decarboxylated to form uridine monophosphate (UMP). It is from UMP that other pyrimidine nucleotides are derived. UMP is phosphorylated to uridine triphosphate (UTP) via two sequential reactions with ATP. Cytidine monophosphate (CMP) is derived from conversion of UTP to cytidine triphosphate (CTP) with subsequent loss of two phosphates
Nucleotides function in cell metabolism Purine ribonucleotides The atoms which are used to build the purine nucleotides come from a variety of sources: The de novo synthesis of purine nucleotides by which these precursors are incorporated into the purine ring, proceeds by a 10 step pathway to the branch point intermediate IMP, the nucleotide of the base hypoxanthine. AMP and GMP are subsequently synthesized from this intermediate via separate, two step each, pathways. Thus purine moieties are initially formed as part of the ribonucleotides rather than as free bases.
Synthesis Purine ribonucleotides By using a variety of isotopically labeled compounds it was demonstrated that the sources of the atoms in purines are as follows: The biosynthetic origins of purine ring atoms N1 arises from the amine group of Asp C2 and C8 originate from formate N3 and N9 are contributed by the amide group of Gln C4, C5 and N7 are derived from Gly - C6 comes from HCO3 (CO2)
DNA is a long polymer made from repeating units called nucleotides.[The DNA chain is 22 to26 Angstroms wide (2.2 to 2.6 nanometres), and one nucleotide unit is 3.3 Ångstroms(0.33 nanometres) long. Although each individual repeating unit is very small, DNA polymers can beenormous molecules containing millions of nucleotides. For instance, the largest humanchromosome, chromosome number 1, is 220 million base pairs long.
Major and minor grooves The double helix is a right-handed spiral. As the DNA strands wind around each other, they leave gaps between each set of phosphate backbones, revealing the sides of the bases inside Two of these grooves twisting around the surface of the double helix: one groove, the major groove, is 22 Å wide and the other, the minor groove, is 12 Å wide. The narrowness of the minor groove means that the edges of the bases are more accessible in the major groove. As a result, proteins like transcription factors that can bind to specific sequences in double- stranded DNA usually make contacts to the sides of the bases exposed in the major groove
Base pairingEach type of base on one strand forms a bond withjust one type of base on the other strand. This iscalled complementary base pairing. Here, purinesform hydrogen bonds to pyrimidines, with Abonding only to T, and C bonding only to G. Thisarrangement of two nucleotides binding togetheracross the double helix is called a base pair. In adouble helix, the two strands are also held togethervia forces generated by the hydrophobic effect and pistacking, which are not influenced by the sequenceof the DNA. As hydrogen bonds are not covalent, theycan be broken and rejoined relatively easily. The twostrands of DNA in a double helix can therefore bepulled apart like a zipper, either by a mechanicalforce or high temperature. As a result of thiscomplementarity, all the information in the double-stranded sequence of a DNA helix is duplicated oneach strand, which is vital in DNA replication.Indeed, this reversible and specific interactionbetween complementary base pairs is critical for allthe functions of DNA in living organisms.
The two types of base pairs form different numbers of hydrogen bonds, AT forming two hydrogen bonds, and GC forming three hydrogen bonds. The GC base pair is therefore stronger than the AT base pair. As a result, it is both the percentage of GC base pairs and the overall length of a DNA double helix that determine the strength of the association between the two strands of DNA.
Long DNA helices with a high GC content have stronger- interacting strands, while short helices with high AT content have weaker-interacting strands. Parts of the DNA double helix that need to separate easily, such as the TATAAT Pribnow box in bacterial promoters, tend to have sequences with a high AT content, making the strands easier to pull apart. Sense and antisense A DNA sequence is called "sense" if its sequence is the same as that of a messenger RNA copy that is translated into protein. The sequence on the opposite strand is complementary to the sense sequence and is therefore called the "antisense" sequence. Since RNA polymerases work by making a complementary copy of their templates, it is this antisense strand that is the template for producing the sense messenger RNA. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands contain both sense and antisense sequences).
Biological molecules that prefer to form strands. Wilkinsworked on the DNA project with Rosalind Franklin, whotook the X-ray photograph that gave Watson and Crick theireureka moment. He then spent almost 10 years rigorouslyverifying that breakthrough. Linking number : in topology, the total number of times one strand of the DNA double helix winds around the other in a right hand direction, given a DNA molecule with constrained ends. 2 molecules differing only in linking number are topoisomers. Writhing number (W) : in topology, the number of superhelical turns in a DNA molecule with constrained ends
Alternative double-helical structures DNA exists in several possible conformations. The conformations so far identified are: A-DNA, B-DNA, C-DNA, D-DNA, E-DNA,H-DNA, L-DNA, P- DNA, and Z-DNA However, only A-DNA, B-DNA, and Z-DNA have been observed in naturally occurring biological systems Which conformation DNA adopts depends on the sequence of the DNA, the amount and direction of supercoiling, chemical modifications of the bases and also solution conditions, such as the concentration of metal ions and polyamines
•The A -DNA is a wider right-handedspiral, with a shallow and wide minorgroove and a narrower and deeper majorgroove. The A form occurs under non-physiological conditions in dehydratedsamples of DNA, while in the cell it may beproduced in hybrid pairings of DNA andRNA strands, as well as in enzyme-DNAcomplexes•B-DNA : the usual double helical structureassumed by double-stranded DNA; seeillustration at deoxyribonucleic acid.•Z-DNA : a form of DNA in which thephosphate groups form a dinucleotiderepeating unit zigzagging up a left-handed helixwith a single, deep groove; it is particularlylikely to occur in stretches of alternating From left to right, the structures of A, Bpurines and pyrimidines and Z DNA
•spacer DNA : the nucleotide sequences occurringbetween genes, in eukaryotes often long andincluding many repetitive sequences; particularly,the DNA occurring between the genes encodingribosomal RNA.•complementary or copy DNA (cDNA) : syntheticDNA transcribed from a specific RNA through thereaction of the enzyme (reverse transcriptase).•nuclear DNA (nDNA) : the DNA of thechromosomes found in the nucleus of a eukaryoticcell.
Repetitive DNA : nucleotide sequences occurring multiply within a genome; they are characteristic of eukaryotes and generally do not encode polypeptides. Sequences may be clustered or dispersed, and repeated moderately (10 to 104 copies per genome) to highly (>106 copies per genome). Moderately repetitive DNA sequences encode some structural genes for ribosomal RNA and histones; highly repetitive sequences are mostly satellite DNA Satellite DNA : short, highly repeated DNA sequences found in eukaryotes, usually in clusters in constitutive heterochromatin and generally not transcribed
Mitochondrial DNA (mtDNA) : the DNA of the mitochondrial chromosome, existing in several thousand copies per cell and inherited exclusively from the mother. Its code differs both from that of nuclear DNA and from that of any present day prokaryote, and it evolves 5 to 10 times more rapidly than nuclear DNA. Recombinant DNA : a DNA molecule composed of linked sequences not normally occurring within the same molecule, such as a bacterial plasmid into which has been inserted a segment of viral DNA. Single copy DNA (scDNA) : nucleotide sequences present once in the haploid genome, as are the majority of the gene sequences encoding polypeptides in eukaryotes
THE CENTRAL DOGMAOF MOLECULAR BIOLOGY ( THE BIOINFORMATION THEORY)
Replication• Replication• Chromosomes are located in the nucleus of a cell. DNA must be duplicated in a process called replication before a cell divides. The replication of DNA allows each daughter cell to contain a full complement of chromosomes.• DNA Replication:• Semiconservative Model of DNA Replication After Watson and Crick proposed the double helix model of DNA, three models for DNA replication were proposed: conservative, semiconservative, and dispersive. The semiconservative model was proved to be the correct one
Semiconservative DNA replicationThe two strands in the double helixseparate, and then each strand serves astemplate for the synthesis of a new(complementary) strand. Afterreplication has been completed, each ofthe two duplexes has one old and onenewly synthesized strand. and dispersive modes of replication donot make much sense, and are notsupported by experiments.
Eukaryotic DNA replication is semiconservative Eukaryotic DNA replicates Semiconservatively by the Taylor, Woods, and Hughes experiment in 1958. They labeled DNA with 3H-T, treated the roots of Fava bean with Colchicin, fixed and prepared for microscopy. At the first metaphase, after labeling at interphase, both chromatids of each chromosome ere labeled, whereas at the second metaphase only one chromatide was labeled
How does this show semiconservative replication?
The DNA double helix and genetic replication• Because an A on one strand must base-pair with a T on the other strand, if the two strands are separated, each single strand can specify the composition of its partner by acting as a template.• The DNA template strand does not carry out any enzymatic reaction but simply allows the replication machinery (an enzyme) to synthesize the complementary strand correctly.• This dual-template mechanism is termed semi- conservative, because each DNA after replication is composed of one parental and one newly synthesized strand. Because the two strands of the DNA double helix are interwound, they also must be separated by the replication machinery to allow synthesis of the new strand. Figure 3 shows this replication.
Features of DNA replication• Bidirectional. Starts at specific sites (origins) and moves• in opposite directions using two replication “forks”.• • Semi-discontinuous. One strand (leading) replicates continuously and the other (lagging) discontinuously• • In the 5’ - 3’ direction. Enzymes (DNA polymerases) can only add a nucleotide to a free OH group at the 3-end of a growing chain
The double-stranded DNA shown above is unwinding and ready forreplication. Note the antiparallel nature of the strands; that is, the 5to 3 orientation of the top strand and the 3 to 5 orientation of thecomplementary bottom strand.A. The DNA is already partially unwound toform a replication fork.B. On the bottom template strand, primasesynthesizes a short RNA primer in the 5 to 3direction.C. Primase leaves, and DNA polymerase addsDNA nucleotides to the RNA primer in the 5to 3 direction. In E. coli the enzyme used isDNA polymerase III. This new DNA is calledthe leading strand because it is being made inthe same direction as the movement of thereplication fork.
Enzymes and Proteins in DNA Replication• A large number of enzymes and other proteins are involved in the synthesis of new DNA at a replication fork.
• Alternative DNA polymerase:• This DNA polymerase replaces the RNA primer with DNA. This is a different type of DNA polymerase from the main DNA polymerase which synthesizes DNA on a DNA template.• In E. coli the main enzyme is DNA polymerase III and the enzyme that replaces the RNA primer with DNA is DNA polymerase I.• When the RNA primer has been replaced with DNA, there is a gap between the two Okazaki fragments and this is sealed by DNA ligase
DNA ligase:• DNA ligase seals the gap left between Okazaki fragments after the primer is removed. As the Okazaki fragments are joined, the new lagging strand becomes longer and longer. • DNA polymerase:• Location: On the template strands.• Function: Synthesizes new DNA in the 5 to 3 direction using the base information on the template strand to specify the nucleotide to insert on the new chain. Also does some proofreading; that is, it checks that the new nucleotide being added to the chain carries the correct base as specified by the template DNA. If an incorrect base pair is formed, DNA polymerase can delete the new nucleotide and try again.
• • Lagging Strand:• The new DNA strand made discontinuously in the direction opposite to the direction in which the replication fork is moving.• • Leading strand:• The new DNA strand made continuously in the same direction as movement of the replication fork.• • Okazaki fragment:• Location: On the template strand which dictates new DNA synthesis away from the direction of replication fork movement.• Function: A building block for DNA synthesis of the lagging strand. On one template strand, DNA polymerase synthesizes new DNA in a direction away from the replication fork movement. Because of this, the new DNA synthesized on that template is made in a discontinuous fashion; each segment is called an Okazaki fragment.
• • Helicase:• Location: At the replication fork.• Function: Unwinds the DNA double helix.• • Primase:• Location: Wherever the synthesis of a new DNA fragment is to commence. Function: DNA polymerase cannot start the synthesis of a new DNA chain, it can only extend a nucleotide chain primer. Primase synthesizes a short RNA chain • Single-strand binding (SSB) proteins: that is used as the primer for DNA Location: On single-stranded DNA synthesis by DNA polymerase. near the replication fork. Function: Binds to single-stranded DNA to make it stable.
• Overall direction ofreplication (movementof replication fork):The direction of replication i.e.,the direction in which thereplication fork moves as theDNA double helix unwinds.• Parent DNA:The parental DNA double helixthat will be unwound and usedas the template for new DNAsynthesis.