Nucleic Acids 2 PDF - Lecture Notes

LECTURE NUCLEIC ACIDS II  DNA Replication Involves duplication of a double stranded DNA molecule (DNA makes a copy of itself). Occurs during interphase stage of cell division called Mitosis. Replication is important so that when the cell divides into two cells each of the d...

LECTURE NUCLEIC ACIDS II  DNA Replication Involves duplication of a double stranded DNA molecule (DNA makes a copy of itself). Occurs during interphase stage of cell division called Mitosis. Replication is important so that when the cell divides into two cells each of the daughter cell must retain a copy of the parent DNA molecule. Genetic information on the DNA molecule has to be retained in its original form during replication.  DNA replication involves several processes: - first, the DNA must be unwound, separating the two strands. - Each strand then act as templates for synthesis of the new strands, which are complimentary in base sequence. - bases are added one at a time until two new DNA strands that exactly duplicate of the original DNA are produced. Semi-Conservative replication process DNA replication is Semi-conservative. The parent DNA strand separates into two Each strand serves as a template for new complementary strands The new double helix is half original because one strand of each daughter DNA comes from the parent DNA and one strand is new Semi-conservative was the accepted model after Meselson and Stahl did experiments (Meselson & Stahl, 1958).  Meselson-Stahl experiment provided evidence and renders conservative and dispersive method of DNA replication redundant. Conservative hypothesis proposed that the entire DNA molecule acted as a template for the synthesis of an entirely new one. The original copy of DNA is left unchanged following synthesis of its new daughter duplicate. Dispersive hypothesis suggested that new DNA molecules were synthesised by breaking the DNA in short pieces alternating from one strand to the other, resulting in two new daugher DNA molecules. Mechanism of DNA replication  Mechanism of DNA replication Enzyme Topoisomerase unknots and uncoils the DNA molecule to reduce the strain of unwinding. Enzyme helicase break the hydrogen bonds to unwind or unzip several sections of parent DNA molecule. The open DNA section is called a replication fork. DNA polymerase (Complex) extends both the leading and lagging strand by adding complimentary nucleotides one by one in a 5’ to 3’ direction.  The templates are all read by DNA polymerase enzymes in a 3’ to 5’ direction On the leading strand DNA polymerase III requires enzyme primase (RNA polymerase) to synthesise an initial RNA primer (short piece of nucleotide). The primer acts as bind site for DNA polymerase III and are then replaces with DNA nucleotides continuously adding them one by synthesizing the Leading strand in a 5’ to 3’ direction.  On the lagging strand primase synthesise RNA primers. The DNA polymerase I replaces the RNA primers with DNA nucleotides in a classic 5’ to 3’ away from the replication fork. DNA polymerase I forms short pieces called okazaki fragments. Okazaki fragments are joined by enzyme DNA ligase which catalyzes the formation of the phosphodiester bond between pieces of DNA. The lagging strand is therefore discontinuously in a reverse fashion.  Genetic Information carried by the DNA is used to synthesise polypeptides (protein) molecules. Protein synthesis occurs in two major stages; 1. Transcription The process by which the DNA genetic code is read and transferred to messenger RNA (mRNA). 2. Translation The process by which the genetic code is converted into a protein (end product of gene expression).  THE GENETIC CODE Genetic code is a sequence of bases on a gene. Gene: sequence of bases (nucleotide) on a DNA coding for a specific amino acid. The genetic code is combination of three different base (nucleotides) in a triplet sequence that codes the 20 amino acids. A triplet of bases along the mRNA that codes for a particular amino acid is called a codon. The genetic code consist of 64 (43)triplet bases sequences or codons.  The codons are written 5'-->3', as they appear in the mRNA. Some codon may signal the “start” and “end” of a polypeptide chain. Codon AUG is an initiation codon and codes for methionine. It signal the start of polypeptide chain. Codon UAA, UAG, and UGA do not code for any amino acid and serve to STOP further addition of amino acid to a growing polypeptide chain mRNA codon and associated amino acids  Degeneracy of the genetic code Some amino acids are represented by more than one codon, meaning that the genetic code is degenerate (redundant). It means the genetic code has more codes than required. For example, four codes (GGU, GGC, GGA and GGG) all translate for Glycine. Many other amino acids in the genetic code have degenerate codes. Except Methionine which is specified by one code (AUG).  Genetic code is degenerate (redundant) but not ambigous. For example codon GAA and GAG both specify glutamic acid (redundancy) but neither of them specify any other amino acids (no ambiguity). Degree of degeneracy can be varies among amino acids. For example glutamic acid exhibits twofold degenerate site.  Guanine exhibits threefold degenerate site; three codes all translate for Guanine Glycine exhibits fourfold degenerate site. Four codes all translate for Glycine. Methionine (AUG) exhibits non degenerate site; Only one code translate for methionine. Note: if a nucleotide base sequence at the 3’ position was changed to any of the four bases (i.e due to mutation), a codon would not specify for the same amino acid.  Reading the Genetic Code Exercise: Suppose we want to determine the amino acids coded for in the following section of a mRNA 5’—CCU —AGC—GGA—CUU—3’ According to the genetic code, the amino acids for these codons are: CCU = Proline AGC = Serine GGA = Glycine CUU = Leucine The mRNA section codes for the amino acid sequence of Pro—Ser—Gly—Leu  Transcription Transcription takes place in a manner similar to DNA replication. The double stranded structure unwinds and one of the strands serves as a template for RNA formation. The unwinding is accomplished DNA helicase which breaks down the hydrogen bonds between nitrogenous bases.  One of the DNA strands in the double helix holds the genetic information used for protein synthesis. This is called the sense strand, (or information strand) and runs in 5’ to 3’ direction. The complementary strand that binds to the sense strand is called the anti-sense strand. The antisense serves as a template for generating a mRNA molecule that delivers a copy of the sense strand information to a ribosome. The antisense strand runs in 3’ to 5’ direction.  Transcription start when RNA polymerase is joined to a protein called sigma which recognises the proper starting point. RNA polymerase binds to a nucleotide sequence in the DNA called a promoter site. RNA polymerase reads the DNA antisense strand in the 3’ to 5’ direction and polymerises mRNA down in a 5’ to 3’ direction.  Three bases on the DNA strand matches with corresponding three bases on the mRNA (codon) For example a triplet of base sequence of ATC on DNA would correspond to codon UAG on mRNA. The messenger molecule is released into the cytoplasm to find a ribosome, and the DNA then rewinds to its double helix structure. mRNA moves out of the nucleus through nuclear pores to the ribosomes where translation takes place.  Translation In the cytoplasm, mRNA travels to ribosomes where it’s genetic material (codon) is decoded (interpreted) to assemble amino acids into protein. This process is called translation. The process is achieved with help of transfer RNA. Before translation, Ribosomal RNA is dissociated into small and large subunits. The initial complex consist of small ribosomal subunits, mRNA and tRNA.  The large ribosomal units consist of an amino acyl site (A) where incoming activated tRNA attaches and peptidyl site (P) where amino acids are joined through peptide bonds. The anticodon of tRNA are used to read the codon on the mRNA. Each condon on mRNA represents an amino acid. In the ribosome the mRNA is read in the same direction it was polymerised in the 5’ to 3’ direction.  Amino acids attach at 3’ binding end of tRNA with aid of Adenosine Triphosphate (ATP) as a source of energy and specific enzyme complexes called aminoacyl-tRNA synthetases. Each of the 20 amino acids has it own type of tRNA to work with in a cell. END OF LECTURE! THANK YOU.