BIOLOGICAL BACKGROUND THE CENTRAL DOGMA OF MOLECULAR BIOLOGY

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1 BIOLOGICAL BACKGROUND Central Dogma DNA and RNA Structure Replication, Transcription and Translation Techniques of Molecular Genetics Using restriction enzymes Using PCR THE CENTRAL DOGMA OF MOLECULAR BIOLOGY Genetic information flow: 1) From DNA to DNA during its transmission from generation to generation. 2) From DNA to Protein during its phenotypic expression in an organism. Transcription: DNA to RNA (sometimes reversible). Translation: RNA to protein (irreversible). Occassionally, genetic information flows from RNA to DNA (reverse transcription).

2 HEREDITARY MATERIAL Major structural differences between DNA and RNA are: 1) Hydrogen vs. Hydroxide 2) Thymine (T) vs. Uracil (U) Most organisms and viruses have DNA as their hereditary material. Some viruses have RNA (single and double stranded). BUILDING BLOCKS DNA molecules are typically composed of two strands that are related through complementary base pairs (Hydrogen bonds). Phosphodiester links: Nucleotides are linked together by phosphodiester backbone. Anti-parallel strands Purines A, G Pyrimidine C, T (U)

3 TYPES OF RNA Four different classes of RNA molecules play essential roles in gene expression: Messenger RNA (mrna): intermediaries that carry genetic information from DNA to the ribosomes where proteins are synthesized. Transfer RNA (trna): small RNA molecules that function as adaptors between amino acids and the codons in mrna during translation. Ribosomal RNA (rrna): structural components of the ribosomes, the intricate machines that translate nucleotide sequences of mrnas into amino acid sequences of polypeptides. Small Nuclear RNA (snrna): structural components of spliceosomes, the nuclear structures that excise introns from nuclear genes. ORGANISMS Organisms (including bacteria and blue-green algae) that lack true nuclei in their cells and that do not undergo meiosis Coupled transcription and translation Organisms that have nuclei enclosed by a membrane within their cells

4 CHROMOSOMES Prokaryotic chromosome: Most contain a single, double stranded, circular DNA molecule. The DNA is mostly naked, but is supercoiled and looped. Eukaryotic chromosome: Consist of very long, linear double stranded DNA. The DNA is complexed with twice as much protein (histones organized in nucleosomes). The protein helps compact it into the nucleus. Region of DNA that controls a hereditary characteristic. It usually corresponds to a sequence used in the production of a specific protein or RNA. GENE A gene carries biological information in a form that must be copied and transmitted from each cell to all its progeny. This includes the entire functional unit: coding DNA sequences, non-coding regulatory DNA sequences, and introns. Genes can be as short as 1000 base pairs or as long as several hundred thousand base pairs.

5 GENE Intron and Exon Exon: segment of a eukaryotic gene that corresponds to the sequences in the final processed RNA transcript of that gene. In some species (including humans) exons are separated by long regions of DNA (introns). Intron: Intervening sequences of DNA bases within eukaryotic genes that are not represented in the mature RNA transcript because they are spliced out of the primary RNA transcript. TRANSFER OF GENETIC INFORMATION Perpetuation of genetic information from generation to generation DNA T T T A A A DNA Replication DNA-dependent DNA polymerase DNA T T T A A A T T T A A A Control of the phenotype: Gene expression Transcription DNA-dependent RNA polymerase mrna Reverse transcription RNA-dependent DNA polymerase (reverse transcriptase) U U U Translation Complex process involving ribosomes, trna and other molecules Polypeptide (not folded) phe

6 REPLICATION Essential components: 1) template 2) dntp 3) DNA polymerase 4) primer To reproduce, a cell must copy and transmit its genetic information (DNA) to all of its progeny. DNA replicates, following the process of semiconservative replication. Each strand of the original molecule acts as a template for the synthesis of a new complementary DNA molecule following the rules of complementary base pairing: adenine (A) to thymine (T) and guanine (G) to cytosine (C). REPLICATION When DNA replicate, many different proteins work together to accomplish the following steps: 1) The two parent strands are unwound with the help of DNA helicases. 2) Single stranded DNA binding proteins attach to the unwound strands, preventing them from winding back together. 3) The strands are held in position, binding easily to DNA polymerase, which catalyzes the elongation of the leading and lagging strands (DNA polymerase also checks the accuracy of its own work). 4) DNA polymerase requires a primer (DNA or RNA). 5) DNA polymerase on the leading strand can operate in a continuous fashion. 6) RNA primer is needed repeatedly on the lagging strand to facilitate synthesis of Okazaki fragments (DNA primase helps to build the primer). 7) Each new Okazaki fragment is attached to the completed portion of the lagging strand in a reaction catalyzed by DNA ligase.

7 REPLICATION - Replicate DNA in the 5 3 direction -Also has 3 5 exonuclease activity that eliminates RNA primers on lagging strand Continuous synthesis Unwinds DNA Discontinuous synthesis Lays down RNA primer TRANSCRIPTION RNA-polymerase-catalyzed synthesis of RNA from a DNA template. Transcription Proceeds in three distinct phases: 1. Initiation (binding of RNA polymerase to template DNA). 2. Elongation (nucleotides are added to the DNA template). 3. Termination (the enzyme and RNA is release from DNA template). Initiation: occurs in a specific region on the DNA called the promoter. The promoter contains sequence elements that bind several transcription factors in combination with RNA polymerase and indicate the first base to be copied into an RNA transcript (the promoter also includes sequences involved in regulation of transcription). Several transcription factors called initiation factors are necessary for RNA polymerases to recognize and bind tightly to the promoter (no primer is necessary). The initiation factors bind first to the promoter, forming an active complex that fits one or more sites on the RNA polymerase enzyme. The enzyme then binds tightly to the initiation factors and the promoter, and the DNA unwinds in their promoter region. Of the two DNA nucleotide chains fully exposed by the unwinding, only one contains the correct promoter sequences and acts as a template. The opposite, non-template chain is complementary to the promoter and template but does not contain encoded information (different genes may have their template chains on either side of the DNA helix).

8 TRANSCRIPTION (continued) Elongation: Once the first base is added, elongation begins and RNA nucleotides add sequentially until the polymerase reaches the end of the template (30 50 nucleotides per second). Termination: Sequences signaling termination also appear in some eukaryotic genes, such as those encoding rrnas. In eukaryotic mrna genes, termination may be coupled to processing reactions rather than occurring in response to specific signal sequences in the DNA. Termination factors contribute to separation of RNA polymerase and elongating factors from the template and release of the transcript in prokaryotes and possibly also in eukaryotes. RNA PROCESSING: PRE-mRNA mrna Primary transcripts produced in the nucleus must undergo processing steps to produce functional RNA molecules for export to the cytosol: 1) Synthesis of the cap: modified G which is attached to the 5 end of the pre-mrna (cap protects the RNA from being degraded by enzymes). 2) Removal of introns: splicing of nuclear transcripts involves particular sequence signals near splice junctions and is conducted by snrna. 3) Synthesis of the poly(a) tail: transcript is cut at a site when completed, and a poly(a) tail is attached to the exposed 3 end. 4) mrna is now exported to the cytosol. In most mammalian cells, only 1% of the DNA sequence is copied into a functional RNA (mrna). 5) The remainder of the original transcript is degraded and the RNA polymerase leaves the DNA.

9 REVERSE TRANSCRIPTION The process of making a segment of DNA from a segment of RNA utilizing a reverse transcriptase enzyme that reads from 3 to 5 (naturally occurs in retroviruses). The newly formed single stranded DNA is complementary, by virtue of the rules of base pairing, to the originally isolated mrna (Complementary DNA - cdna). cdna represents only the exons or coding region of the gene. A poly dt linker-primer is often used for reverse transcription. mrna G U A A U C C U C cdna Reverse transcriptase T T A G G A G C A T T A G G A G C A T T A G G A G C A T A G G A G C A T A G G A G C A T A G G A G C C A A T T A T A G G G A A G G C A T T A G G A G The ribosome binds to the mrna at the start codon (AUG) that is recognized only by the initiator trna. The ribosome proceeds to the elongation phase of protein synthesis. Complexes, composed of an amino acid linked to trna, sequentially bind to the appropriate codon in mrna by forming complementary base pairs with the trna anticodon. The ribosome moves from codon to codon along the mrna. Amino acids are added one by one, translated into polypeptidic sequences dictated by DNA and represented by mrna. At the end, a release factor binds to the stop codon, terminating translation and releasing the complete polypeptide from the ribosome. TRANSLATION

10 THE GENETIC CODE One specific amino acid can correspond to more than one codon. Key Ala Phe Lys Pro Thr Cys Gly Leu Gln Val Amino Acid Alanine Phenylalanine Lysine Proline Threonine Cysteine Glycine Leucine Glutamine Valine Key Asp His Met Arg Trp Glu Ile Asn Ser Tyr Amino Acid Aspartic acid Histidine Methionine Arginine Tryptophane Glutamic acid Isoleucine Asparagine Serine Tyrosisne CONTROL OF GENE EXPRESSION Examples of regulation at each of the steps are known, although for most genes the main site of control is step 1: transcription of a DNA sequence into RNA

11 TECHNIQUES OF MOLECULAR GENETICS The action of restriction enzymes: Restriction enzymes (EcoRI in this example), surrounds the DNA molecule at the point it seeks(sequence GAATTC). It cuts one strand of the DNA double helix at one point and the second strand at a different, complementary point (between the G and the A base). The separated pieces have single stranded "sticky-ends," which allow the complementary pieces to combine. The newly joined pieces are stabilized by DNA ligase. SOUTHERN BLOTTING Detection of specific DNA fragments by gel-transfer hybridization 1) The mixture of double-stranded DNA fragments generated by restriction nuclease treatment of DNA is separated according to length by electrophoresis. 2) A sheet of either nitrocellulose paper or nylon paper is laid over the gel, and the separated DNA fragments are transferred to the sheet by blotting. 3) The gel is supported on a layer of sponge in a bath of alkali solution, and the buffer is sucked through the gel and the nitrocellulose paper by paper towels stacked on top of the nitrocellulose. 4) As the buffer is sucked through, it denatures the DNA and transfers the single-stranded fragments from the gel to the surface of the nitrocellulose sheet, where they adhere firmly. (This transfer is necessary to keep the DNA firmly in place while the hybridization procedure is carried out). 5) The nitrocellulose sheet is carefully peeled off the gel. 6) The sheet containing the bound single-stranded DNA fragments is placed in a sealed container together with buffer containing a radioactively labeled DNA probe specific for the required DNA sequence. 7) The sheet is exposed for a prolonged period to the probe under conditions favoring hybridization. 8) The sheet is removed from the container and washed thoroughly, so that only probe molecules that have hybridized to the DNA on the paper remain attached. 9) After autoradiography, the DNA that has hybridized to the labeled probe will show up as bands on the autoradiograph.

12 SOUTHERN BLOTTING An adaptation of this technique to detect specific sequences in RNA is called Northern blotting. In this case mrna molecules are electrophoresed through the gel and the probe is usually a single-stranded DNA molecule. Northern blots allow investigators to determine the molecular weight of an mrna and to measure relative amounts of the mrna present in different samples. INSERTING DNA INTO A PLASMID Plasmid vectors are small circular molecules of double stranded DNA derived from natural plasmids that occur in bacterial cells. A piece of DNA can be inserted into a plasmid if both the circular plasmid and the source of DNA have recognition sites for the same restriction endonuclease. The plasmid and the foreign DNA are cut by this restriction endonuclease producing intermediates with sticky and complementary ends. Those two intermediates recombine by basepairing and are linked by the action of DNA ligase. Few mismatches occur, producing an undesirable recombinant. The new plasmid can be introduced into bacterial cells that can produce many copies of the inserted DNA.

13 CLONING INTO A PLASMID Plasmid is used to import recombinant DNA into a host cell for cloning. DNA fragment that contains a gene of interest is inserted into a cloning vector or plasmid. The plasmid carrying genes for antibiotic resistance, and a DNA strand, which contains the gene of interest, are both cut with the same restriction endonuclease. The plasmid is opened up and the gene is freed from its parent DNA strand. They have complementary "sticky ends." The opened plasmid and the freed gene are mixed with DNA ligase, which reforms the two pieces as recombinant DNA. Plasmids + copies of the DNA fragment produce quantities of recombinant DNA. This recombinant DNA stew is allowed to transform a bacterial culture, which is then exposed to antibiotics. All the cells except those which have been encoded by the plasmid DNA recombinant are killed, leaving a cell culture containing the desired recombinant DNA. DNA cloning allows a copy of any specific part of a DNA (or RNA). This technique is the first stage of most of the genetic engineering experiments: production of DNA libraries, PCR, DNA sequencing, etc. CONSTRUCTION OF GENOMIC or cdna LIBRARY A genomic library comprises a set of bacteria, each carrying a different small fragment of genomic DNA or cdna. For simplicity, cloning of just a few representative fragments (colored) is shown. In reality, all the gray DNA fragments will also be cloned.

14 SCREENING A GENOMIC or cdna LIBRARY DNA probes or antibodies are used to screen libraries for specific DNA sequences: Transform E. Coli with a genomic library. Plate on selective growth medium; colonies grow. Replica plate colonies transfer onto new selective medium plate with membrane filter or surface; colonies grow on filter. Filter removed from culture dish, bacteria lysed, DNA denatured and bound to filter. Probe DNA hybridized to DNA on filter. Wash filter free of unbound probe. Detect hybridization by autoradiography for radioactively labeled probes. PCR

15 QUANTITATIVE RT-PCR Real-time reverse-transcriptase (RT) PCR quantifies the initial amount of the template. Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle (i.e., in real time) as opposed to the endpoint detection by conventional quantitative PCR methods. The real-time PCR system is based on the detection and quantification of a fluorescent reporter. This signal increases in direct proportion to the amount of PCR product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. REFERENCES Principles of Genetics. Ed.: D. P. Snustad, M. J. Simmons and J. B. Jenkins. John Wiley & Sons, Inc., New York