DNA replication DNA must replicate before a cell divides so that each daughter cell inherits a copy of each

gene. A cell that has no nucleus (that carries gene) or a cell that miss a critical gene are bound to die, just like an individual that has genetic disease may die early. Thus it is essential that the process of the original genetic information produces and absolutely accurate copy of the original genetic information. If mistakes are made in critical genes may result to lethal mutations. The structure of the DNA molecule suggested the mechanism for its accurate replication. DNA replication is a Semiconservative replication w/c demonstrates that each parental strand of the DNA molecule serves as the template for the synthesis of a new daughter strand and that each newly synthesized DNA molecule is composed of one parental strand and one newly synthezied daughter strand. Bacterial DNA replication bacterial chromosomes is a circular molecule of DNA made up of about 3milllion nucleotides DNA replication begins at a unique sequence on the circular chromosome known as replication origin. Replication occurs bidirectionally at the rate of about 500 new nucleotide every second. The point at w/c the new deoxyribonucleotide is added to the growing daughter strand is call replication fork. it is here that the DNA has been opened to allow binding of the various proteins and enzymes responsible for DNA replication Since DNA synthesis occurs bidirectionally, there are two replication fork moving in opposite directions. Replication completes when replication forks collide approximately halfway around the circular chromosome. Functions of key proteins involved in DNA replication. DNA helicase breaks hydrogen bonds between dna strands.

Topoisomerase alleviates positive suppercoiling. Single-strand binding proteins keep the parental strand apart. Primase synthesize RNA primer. DNA polymerase III synthesizes a daughter strand of DNA. DNA polymerase I excises the RNA primers and fills in with DNA DNA ligase covalently links the DNA fragments together. Two different mechanism for the replication of the twon strands. Leading replicated continuously Lagging strand replicated discontinously

Eukaryotic DNA replication DNA replication of eukaryotic is more complex. Human genome consists of approximately 3billion nucleotide pairs. DNA replication begins at many replication origins and proceeds bidirectionally along each chromosome. Information flow in biological system The Central Dogma of genetics is: DNA is transcribed to RNA which is translated to protein. Protein is never back-translated to RNA or DNA; and except for retroviruses, DNA is never created from RNA. Furthermore, DNA is never directly translated to protein. DNA to RNA to protein. DNA, RNA, and Proteins
DNA RNA

is long-term storage. It is stable, packaged, and inert.

is short-term storage. It is unstable and lacks secondary structure. Some RNA has enzymatic activity.
Proteins

are the 'programs' of the cells. They are the physical manifestations of the abstract information recorded in the genome.
Transcription is the process of creating RNA from DNA.

Transcription occurs in the cell's nucleus.

 RNA polymerase is the protein molecule that reads the DNA and creates the RNA
intermediary.

Transcription requires: DNA, RNA polymerase, ribonucleotides, and some ATP for
energy.

Uracil (U) is substituted for thymine (T) in RNA. Transcription initiation is the main point of regulation of gene expression.

A. Initiation: RNA polymerase is an enzyme complex which:

unwinds and unzips DNA double strand attaches to promoter region of gene, which marks the beginning point for transcription

B. Elongation: RNA polymerase:

uses DNA anti-sense strand as a template synthesizes a complementary RNA strand using base pairing rules

C. Termination: RNA polymerase :

A = U

T = A

G = C

C = G

reaches termination region of the gene, which marks the end of the coding sequence terminates transcription by releasing both DNA and RNA

post transcriptional process of RNA prokaryotes : termination releases a mature mRMA for translation. Because bacteria have no true membrane separating the DNA from the cytoplasm, translations begins long before the mRNA is completed. Eukaryotes : transcription produces a primary transcript that must undergo extensive posttranscriptional modifications before it is exported out of the nucleus for translation in the cytoplasm. 3 post-transcriptional modifications : 1st modification - 5 cap structure a cap structure is enzymatically added to the 5end of the primary transcript The cap structure consists of 7 methyl-guanosine attached to the 5 of the RNAby 5 by 5 triphosphate bridge. The 1st 2 nucleotides are also methylated. The cap structure is required for efficient translation of the final mature mRNA 2nd modification - 3 poly(A) tail

the enzymatic addition of a poly (A) tail to the 3end of the transcript. Poly(A) polymerase uses ATP and catalyzes the stepwise polymerization of 100-200 adenosine nucleotides on the 3end of the RNA. Poly (A) tail protects the 3 end of the mRNA from enzymatic degeneration and thus prolongs the lifetime of the mRNA. 3rd modification - RNA splicing Involves the removal of portions of the primary transcript that are not protein coding. Primary transcripts contains both the introns and exons Introns noncoding and intervening sequences Exons protein coding sequences Genetic code the mRNA carries the genetic code for a protein which is triplet code. Since there are only 4 letter in the DNA alphabet ( A, T, G, & C) and because there are 20 amino acids, the genetic code must contain words made of at least 3 letters from 4 letter in the DNA alphabet. Mutations were introduced into the DNA of bacterial virus. These mutations inserted or deleted 1, 2, or 3 nucleotides into/from a gene. When 1 or 2 nucleotides were inserted, no protein was produced. However when a 3rd base was inserted, the sense of the mRNA was restored and the protein was made. Experiment used a sentence of only 3-letter words, inserting 1, 2, and 3 letters and see how that restored the sentence reading frame. THE CAT RAN OUT. THE FCA TRA NOU T. THE FAC ATR ANO UT. THE FAT CAT RAN OUT. Codon - Each group of 3 nucleotides in the sequence of the mRNA and each codon codes for a single amino acids. If sequence is interrupted or changed, it can change the amino acid composition of protein that is produced or even result in the production of no protein at all. A 3-letter genetic code is degenerate code that contains 64 codons and 3 of the codons specify termination signals for the process of translation while triplet codons may serve as code words for same amino acids. Protein sysnthesis : The process of protein synthesis is translation and it is carried out on the ribosomes, which are complexes of ribosomal RNA (rRNA) and proteins.

2 ribosomal sub-units : Small ribosomal sub-unit contains one rRNA molecule and 33 different ribosomal proteins Large ribosomal sub-unit contains 3 rRNA molecules and 49 different ribosomal proteins The molecule that decodes the information in the mRNA molecule into the primary structure of a protein is transfer RNA (tRNA) Protein synthesis involves the simultaneous action of many ribosomes on a single mRNA molecule. The complexes of many ribosomes along a single mRNA are known polyribosomes or polysomes. Each ribosome is synthesizing one copy of the protein molecule encoded by the mRMA. Thus many copies of protein are simultaneously produced.

The Role of the Transfer RNA, tRNA To decode the genetic message into the primary sequence of a protein, the tRNA must perform two functions : 1st, the tRNA must covalently bind one, and only one, specific amino acid. There is at least one tRNA for each amino acid. All tRNA must have a sequence CCA at their 3 ends. This is the site where the amino acids will covalently attached to the tRNA molecule. Each tRNA is specially recognized by the active site of an enzyme called aminoacyl tRNA synthetase that recognizes the correct amino acid and covalently link amino acid to the 3 end of the tRNA molecule to form a structure called aminoacyl tRNA. The covalently bound amino acid will be transferred from tRNA to a growing polypeptide chain during protein synthesis. 2nd, the tRNA must be able to recognize the appropriate codon on the mRNA that calls for that amino acid. This is mediated thru a sequence of 3 base anticodon, which is located at the bottom of the tRNA cloverleaf. The anticodon sequence for each tRNA is complementary to the codon on the mRNA that specifies a particular amino acid. . the complementary hydrogen bonding will bring the correct amino acid to the site of protein synthesis. Process of translation initiation The 1st stage of protein synthesis is initiation. Initiation factors are proteins that assist in the formation of a translation complex composed of an mRNA molecule, small and large ribosomal subunits of ribosome and the initiator tRNA. This initiator tRNA recognizes the codon AUG and carries the amino acid methionine The ribosomes has 2 site of binding tRNA molecules : 1st site, peptidyl tRNAbinding site(P-site), holds the peptidyl tRNA, the growing peptide bound to a tRNA molecule.

2nd site, aminoacyl tRNA biding site, holds the aminoacyl tRNA carrying the next amino acid to be added to the peptide chain. Each of the tRNA molecule is hydrogen bonded to the mRNA molecule by condon-anticodon complementary. The entire complex is further stabilized by the fact that the mRNA is also bound to the ribosome. The initiator methionyl tRNA occupies the P-site in this complex. Chain elongation 2nd stage of translation is chain elongation. 3 steps that are repeated until protein synthesis is completed. 1st step, binding of the aminoacyl-tRNA molecule to the empty A-site. 2nd step, peptide bond formation occurs. This is catalyzed by an enzyme on the ribosome called peptidyl transferase. Now peptide chain is shifted to the tRNA that occupies the A-site. 3rd step, Finally the tRNA in the P-site falls away and the ribosomes changes positions so that the next codon on the mRNA occupies the A-site. This movement of the ribosome is translocation. The process shifts the new peptidyl tRNA from A-site to the P-site. The chain of elongation stage of translation requires the hydrolysis of GPT to GDP and P. several elongation factors are also involve in this process. Termination The last stage of translation is termination. 3 termination codons : UAA, UAG, and UGA For which there is no corresponding tRNA molecules. When one of these stop, codon is encountered, translation is terminated. A release factor binds the empty A-site. The peptidyl transferase that had previously catalyzed peptide bond formation hydrolyzes the ester bond between the peptidyl tRNA and the last amino acid of the newly synthesized protein. At this point the tRNA, the newly synthesized peptide, and the 2 ribosomal subunits are released. Peptide that are released following translation is not necessarily in its final functional form. In some cases the petide is proteolytically cleaved before it becomes functional. Synthesis of digestive enzymes uses this strategy. Sometimes protein must associate with other peptides to form a functional protein as in the case of hemoglobin. These final modifications are specific for particular proteins and like the sequence of the protein itself are directed by the cellular genetic information.

Mutations Mutation is a genetic change that occurs in the nucleotide chain of a DNA molecule. Factors : Mistakes made by DNA polymerase during DNA replication. Result from the action of chemical calles mutagens, that damage the DNA. Mutation Classifications thru changes : Point mutation, substitution of a single nucleotide for another. Ie. Normal DNA sequence : ATGGACTTC Point mutation : ATGCACTTC Deletion mutation, a single nucleotide or even large section of DNA are lost. ie. Normal DNA sequence : ATGGACTTC Deletion mutation : ATGTTC Insertion mutation, one or more nucleotides are added to a DNA sequence. ie. Normal DNA sequence : ATGGACTTC Insertion mutation : ATGCTCGACTTC RESULT OF MUTAIONS Silent mutations DNA changes do not cause changes in the protein, howeven mutation has negative effects on the body and effects depends on how ot alters the genetic code for a protein. Not silent mutaions DNA changes in the codon the result to incorporation of wrong amino acid into the protein, casuing protein to be nonfunctional or to function improperly. Mutagens and carcinogens Mutagens are chemicals that cause a change in the DNA sequence. Mutagens are also carcinogens, cancer-causing chemicals. Most cancel results from mutations in one single normal cell. These mutations result in the loss of normal growth control, causing the

abnormal cells to proliferate. If that growth is not controlled or destroyed, it will result death. ie. Cigarette smoke has about 3000 chemical components that are potent mutagens as a result people who smoke have much greater chance of lung cancer than those who dont. UV UV light is another agent that causes damage to DNA. Absorption of UV light by DNA causes adjacent pyrimidines bases to become covalently linked. The product is call pyrimidine dimmer. As a result of pyramidine dimmer formation, there is no hydrogen bonding between these pyramidine molecules and the complementary bases on the other DNA strand. This stretched of DNA cannot be replicated or transcribed. Bacteria Escherichia coli have 4 different mechanism to repair uv light damage. However repair process can make a mistake. Mutations occur when UV damage repair system makes an error and causes a change in the nucleotide sequence of the DNA. DNA repair defects Human repai sys for pyrimidine dimmers is quite complex that it requires 5 enzymes. The enzymes that performs repair is called repair endonucleate that cleavage the sugarphosphate backbone of the DNA near the site of damage. But if the gene encoding this enzyme is defective, pyrimidine dimmers cant be repaired. The accumulation of mutations combines with simultaneous decrease in the efficiency of DNA repair mechanism leads to an increased incidence of cancer such us genetic skin disorder called xeroderma pigmentosum. Recombinant DNA Several tools are required for genetic engineering, including restriction enzymes, agarose gel electrophoresis, hybridization, and cloning vectors. Cloning a DNA fragments involves digestions of the target and vector DNA with a restriction enzymes. DNA ligase joins the target and vector DNA covalently, and the recombinant DNA molecules are introduced into the bacterial cells by transformation. The desired clone is located by using antibiotic selection and hybridization. Many eukaryotic genes have been clone for the purpose of producing medically important proteins. Polymerase chain reaction Using a heat-stable DNA polymerase produced by the bacterium Thermus aquaticus and specific DNA primers, polymerase chain reaction allows the amplification of DNA sequences that are present in small quantities. Thit techniques is useful in genetic screening, dagnosis of viral or bacterial disease, and forensic science.

Human Genome Project The human genome project has identified and mapped the genes of the human genome and determined the complete DNA sequence of each of the chromosomes. To do this genomic libraries were generated and the DNA sequences of the clone were determined. To map the sequences along each chromosome, chromosome walking was used. DNA sequencing involves reactions in which DNA polymerase copies specific DNA seqeunces. Nucleotide analogue that causes chain termination (dideoxynucleotides) are incorporated randomly into the growing DNA chain. This generates a family of DNA fragments that differ in size by one nucleotide. DNA sequencing gels separate these fragments and provide DNA sequence data.