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Lec1: microbial genetics Ass.Prof. Dr. maysa salih Genetics: Is the study of the inheritance or heredity of living things A genome is a complete set of genetic material (genes) in a cell. For example, an ovum or a sperm contains a genome. The genetic material of living things is predominantly DNA. However, the genetic material of some viruses such as HIV and Ebola is RNA. A gene can be defined as a sequence of DNA which codes for the synthesis of one polypeptide. Bacterial chromosome Consists of a single molecule of double-stranded deoxyribonucleic acid (DNA) in ring shape which is in association with histone like proteins. A bacterium contains one chromosome. It contains hereditary information which is passed from one generation to the next generation. The procaryotic chromosome is not surrounded by a nuclear membrane. A bacterium may contain one or more extra piece of chromosomes called plasmids. Plasmids are circular,double-stranded DNA. Plasmids may contain genes responsible for antibiotic resistance. They have been used as vectors to transfer the foreign genes into the bacterial cells in genetic engineering techniques. DNA consists of building blocks called nucleotides. A nucleotide consists of a phosphate, a sugar (deoxyribose) and a nitrogenous base (adenine, guanine, cytosine and thymine). Nucleotides link together to form a polynucleotide (strand).. A DNA consists of two strands arranged in an anti-paralleled direction. If one strand goes from the 3' to 5' direction, the other strand always goes in the 5' to 3' direction. These two strands are held together by hydrogen bonds between their nitrogenous bases according to the base-pairing rules: adenine with thymine, and cytosine with guanine. The number of hydrogen bonds between adenine and thymine is 2, and that between cytosine and guanine is 3. DNA REPLICATION: DNA replication is the process in which DNA is duplicated. In E. coli, the process takes about 20 minutes The Overall Replication Process: The process involves the following basic steps: (1) uncoiling of DNA, (2) unzipping of the hydrogen bonds so each strand serves as a template for the synthesis of a new strand, and (3)synthesis of a new strand whose bases are complementary to those of the old strand.DNA synthesis is described as semiconservative type because each old strand is used as a template for the synthesis of a new strand. So, in every DNA molecule one strand is always an old one and another strand is always a new one. The first stage of DNA replication in prokaryotes is the uncoiling of the DNA double helix by the enzyme helicase. Helicase separates the DNA into two template strands. RNA primase then adds a short sequence of RNA to the template strands. This short sequence of RNA is a primer which allows DNA polymerase III to bind to the strands and start the replication process. Once this is done, DNA polymerase III adds nucleotides to each template strand in a 5'→3' direction. The nucleotides have 3 phosphate groups and are called deoxyribonucleoside triphosphates. Two of these phosphate groups break off during the replication process to release energy. Since the strands are anti-parallel (the two strands have their 5' end and 3' end in opposite sides) and replication can only occur in a 5'→3' direction, one of the strands will be replicated in the same direction as the replication fork and the other will be replicated in the opposite direction of the replication fork. This means that one of the strands is synthesised in a continuous manner (named the leading strand) while the other one is synthesised in fragments (named the lagging strand). The leading strand only needs one primer while the lagging strand needs quite a few as it is formed in fragments. These fragments are called Okazaki fragments. DNA polymerase I will remove the RNA primers and replace these with DNA. The enzyme DNA ligase then joins the Okazaki fragments together to form a continuous strand. TRANSCRIPTION: THE FIRST STAGE OF GENE EXPRESSION Only one strand of DNA is transcribed into mRNA. The strand that contains base sequence for synthesis of protein is called the sense strand (template strand). The strand that does not contain information for the synthesis of protein is called the nonsense, noncoding or antisense strand PROTEIN SYNTHESIS Transcription Translation DNA ------------------ > mRNA ----------------- > Protein The segment of DNA that contains the information for the synthesis of a protein is called a gene. Transcription: The process of synthesizing RNA based on the sequence of nucleotides in DNA is called transcription. It involves three phases: initiation, elongation and termination. Initiation: The initiation of transcription begins with the attachment of RNA polymerase on specific locations (sequences) on DNA molecule called promoters. The promoters determine which DNA strand (sense strand) will be transcribed. The enzyme contains a subunit called the sigma factor (a protein). The sigma factor determines which promoter site on DNA will begin transcription. Once the core of RNA polymerase attaches to the promoter site, the sigma factor dissociates from the enzyme. In most cases, the triplet on DNA that begins transcription is TAC which is transcribed as AUG (codes for methionine) on mRNA. Elongation: The segment of DNA that contains genetic information for a specific protein is uncoupled by RNA polymerase. Free ribonucleotides (ATP, UTP, CTP and GTP) are automatically attracted by the exposed bases on one polynucleotide strand (the sense strand) which contains the genetic information. New nucleotides are linked to the exposed bases by hydrogen bonds according to the base-pairing rules: adenine pairs with uracil, AU; thymine pairs with adenine, T-A; and cytosine pairs with guanine, C-G. The RNA polymerase catalyzes condensation reaction between the newly attached nucleotides by the elimination of inorganic pyrophosphate, forming a new polyribonucleotide which is called messenger -RNA (mRNA). The direction of RNA synthesis is similar to that of DNA, starting from the 5' end to the 3' end of the mRNA. As the mRNA is still being synthesized, its 5' end detaches from the DNA and immediately attaches to the ribosome to initiate translation. In eucaryotes, the mRNA has to move out the nucleus through nuclear membrane before translation can begin. When the mRNA breaks away from the DNA, the two polynucleotide strands of DNA rejoin together. Termination: A specific molecule called the rho protein moves along the sense strand. When it encounters the termination sequence, it signals to the RNA polymerase to end the transcription process. The mRNA then breaks away from the DNA Translation involves three steps: 1. Initiation 2. Elongation 3. Termination Initiation: 5’ end of mRNA binds to the small subunit of the ribosome initial mRNA codon = AUG = start codon tRNA with anticodon: UAC binds to mRNA AUG codon by complementary base pairing, methionine attached to tRNA 3’ terminal large ribosomal subunit binds, completing ribosomal structure, and producing two ribosomal binds sites: P site & A site B. Elongation: tRNA with anticodon complementary to second mRNA codon binds to ribosomal A site, with appropriate amino acid attached to tRNA 3’ terminal enzymes in ribosome catalyze formation of peptide bond between methionine and 2nd amino acid P site tRNA, now separated from methionine, exits ribosome ribosome moves one codon (3 nucleotides) toward the 3’ end of mRNA, thus shifting previous A-site tRNA to P-site, and opening A-site tRNA with anticodon complementary to A-site mRNA codon binds to ribosomal A-site, with appropriate amino acid attached to tRNA 3’ terminal enzymes in ribosome catalyze formation of peptide bond between 2nd and 3rd amino acids P site tRNA, now separated from its amino acid, exits ribosome ribosome moves one codon (3 nucleotides) toward the 3’ end of mRNA, thus shifting previous A-site tRNA to P-site, and opening A-site repetition of process until stop codon is reached C. Termination: when ribosomal A-site reaches a stop codon, no tRNA has a complementary anticodon release factor protein binds to ribosomal A-site stop codon polypeptide and mRNA are released large and small ribosomal subunits separate