Biochemistry Topics
DNA and RNA
DNA is a polymer of deoxyribonucleotide units. The backbone of a strand consists of the deoxyriboses linked by phosphate groups. The 3'-OH of one sugar is joined to the 5'-OH of the next by a phosphodiester bond. This arrangement gives polarity to the strand: one end has a free 5'-OH and the other a free 3'-OH.
Two strands are held together
by hydrogen bonds between the base pairs. Thymine (T) always pairs with adenine
(A) with two hydrogen bonds. Cytosine (C) always pairs with guanine (G) with
three hydrogen bonds. Two strands are coiled around each other, each running
in the opposite direction. The bases are on the inside of the resulting helix.
There are a few possible conformations of the DNA helix, but the most common is the B form. In this conformation, the double helix is right handed with 9.7-10 base pairs per turn. The B form helix forms alternative major and minor groves in the sugar phosphate backbone.
RNA is a polymer of ribonucleotide units, very similar to DNA. The sugar units in RNA are riboses rather than deoxyriboses. One of the bases in RNA is uracil instead of thymine. RNA usually exists as a single-stranded molecule, althought it may form a double helix (not a B form, but a modified A form helix). There are four major classes of RNA: ribosomal, transfer, messenger and small nuclear RNA.
Ribosomal RNA (rRNA) is a major component of ribosomes, which play both catalytic and structural soles in protein synthesis. A prokaryotic ribosome contains three RNA subunits: 5s, 16s and 23s. Eukaryotic ribosomes contain four subunits: 5s, 5,85s, 18s and 28s.
Transfer RNA (tRNA) is the adapter molecule that carries activated amino acids to the ribosome for protein synthesis, in a sequence determined by the mRNA template.
Messenger RNA (mRNA) is linear, single-stranded RNA that contains the code for specific protein synthesis. Small nuclear RNA (snRNA) functions in RNA splicing and protein targeting.
Advance Topics: Cellular and Molecular Biology (1)
DNA
Properties
The strands of DNA helix will come apart if the hydrogen bonds between bases are disrupted by heating. This called "melting" because it occurs abruptly at a given melting temperature (Tm), defined as the temperature at which half the helical structure is lost. The abrupt transition occurs because the hydrogen bonds form a highly cooperative structure, held together by many reinforcing bonds. The more bonds are broken, the more bonds will be easier to break. DNA molecules rich in G-C pairs have higher melting temperatures because G-C forms three hydrogen bonds while A-T forms only two. The A-T regions of a DNA molecule are the first to melt.
In order to fit inside the nucleus, the circular DNA of prokaryotes exist as supercoiled, compact structures. Eukaryotic DNA is linear, and to save space it is bound to small proteins called histones, which form nuclei around which DNA is wound. 140 base pairs are wound around a histone octamer, forming a nucleosome.
The entire DNA-protein complex of eukaryotes, made up of repeating nucleosomes is the chromatin. Heterochromatin is very highly condensed, transcriptionally inactive and makes up about 10% of the chromatin in the non-dividing cell. The rest is euchromatin: decondensed and distributed throughout the nucleus. About 10% of the euchromatin is transcriptionally active, in a more decondensed state that allows transcription.
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Advance Topics: Topological
Properties of DNA (Cellular and Molecular Biology)
DNA Melting and Renaturing (Cellular
and Molecular Biology)
During DNA replication, the strands of a parent molecule are separated and each is used as a template. One of the strands of each daughter DNA molecule is newly synthesized, while the other is passed unchanged from the parent DNA molecule.
DNA polymerases catalyze the formation of phosphodiester bonds during DNA chain elongation. They add deoxyribonucleotides (dNTPs) to the 3'-OH terminus of a preexisting DNA chain or primer. Some polumerases also have a proofreading fiunction: 3' to 5' exonuclease activity removes mispaired nucleotides that were just added, before adding the next.
Fundamental requirements for DNA replication include:
In retroviruses, information flows from RNA to DNA. DNA complementary to viral RNA is synthesized by reverse transcriptase, an enzyme brought into the host cell by the infecting virus. Reverse transcriptase catalyses the synthesis of DNA, the digestion of the RNA template, and the synthesis of the complimentary DNA strand to form a double helix.
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Advance Topics: DNA Replication (Cellular and Molecular Biology)
DNA Polymerases
DNA
polymerase I is a 103 kD monomer that catalyzes the addition of dNTP units to
the 3' end of a DNA chain or primer, usually up to 20 units (moderately possesive),
at a speed of about 10 dNTPs per second. In addition to the polymerase activity
sites, DNA polymerase I contains another two active sites with exonuclease activity.
The 3' to 5' exonuclease activity catalyzes the hydrolysis of unpaired nucleotides,
one at a time, at the 3' end of DNA chains, proofreading the last nucleotide
before addind another. The 5' to 3' exonuclease activity catalyzes the hydrolysis
of the RNA primer and corrects some damage at the 5' terminus or several residues
away.
DNA polymerase III holoenzyme synthesizes most new DNA as part of a multisubunit assembly, while polymerase I erases the primer and fills in the gaps. The holoenzyme catalyzes the rapid formation of mny thousanda of phosphodiester bonds before releasing the tempate. It is designed to grasp the template by means of a hollow beta2 subunit which encircles the template and acts as a sliding clamp.
The holoenzyme consists of 10 kinds of polypeptides and has a mass of about 900 kD. It is a dimer because both strands of parental DNA must be replicated in the same place at the same time. Since the leading and lagging strands are synthesized differently, the holoenzyme is asymetric.
DNA polymerase II participates in repair but is not needed in replication.
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Advance Topics: DNA Replication (Cellular and Molecular Biology)
Replication Fork
Supercoiled DNA molecules
have a linking number that describes their topological arrangement. DNA molecules
differing only in linking number are topoisomers and can be inter-converted
by cutting one or both DNA strands and then rejoining them in an alternative
arrangement. That process is catalyzed by topoisomerases. The relaxation of
supercoiled DNA molecules is catalyzed by topoisomerases in preparation for
DNA replication.
Chromosomes contain unique origins of replication (ori), which are tandem arrays rich in A-T, facilitating melting. The binding of a protein called dnaA near the ori initiates an intricate series of steps leading to the unwinding of the template. Proteins called dnaB and dnaC bind to dnaA. dnaB is an helicase, which catalyses the ATP-driven unwinding of the helical DNA. The unwound portion is the stabilized by single strand binding proteins (SSBs).
A specialized RNA polymerase known as primase joins the dnaABC complex forming a multisubunit assembly called primosome. Primase synthesizes short strands of RNA (~ 5 rNTP) complementary to each of the DNA strands.
Now the DNA strands are ready for DNA polymerases to start replication. A structure known as the replication fork is formed. Both new strands of DNA are synthesized in the 5' to 3' direction. A leading strand is synthesized continuously, while the lagging strand is synthesized in the form of short fragments, known as Okazaki fragments, which are later joined by DNA ligase.
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