3. Translation. 2. Transcription. 1. Replication. and functioning through their expression in. Genes are units perpetuating themselves

Transcription

1 Central Dogma Genes are units perpetuating themselves and functioning through their expression in the form of proteins 1 DNA RNA Protein Replication 2. Transcription 3. Translation Spring

2 Terminology Replication: the process by which the genome is replicated Transcription: the process by which RNA is synthesized Translation: the process by which proteins are synthesized in an RNA template Nucleic acids display directionality: they are usually written 5 -> 3 DNA coding strand: the DNA sequence that bears the same sequence as the mrna (except T-> U) - also known as the sense strand DNA non-coding strand: the DNA strand that directs synthesis of the mrna via a base-pairing mechanism - also known as the anti-sense strand RNA plus strand: the RNA strand with the same polarity as mrna Spring

3 Transcription mrna is the product of transcription: 1) synthesized as a precursor in the nucleus 2) goes through a maturation process in the nucleus 3) mature mrna is transported to cytoplasm 4) 1 mrna = 1 protein species (monocistronic) 5) mrna is synthesized by pol II Features of a eukaryotic mrna: 1) most have a 3 poly A tail (~200nt) 2) methylated 5 cap 3) usually contain introns and exons Transcription can be divided into 3 stages: 1) initiation - RNA polymerase binds to the DNA template. Local DNA denaturation occurs 2) elongation - ribonucleotides are covalently added to the 3 end of a growing RNA molecule 3) termination - endpoint of transcription. A completed RNA molecule is released What are the components involved in transcription? 1) DNA-dependent RNA polymerase - the complex apparatus that is responsible for transcription: the ability to catalyze RNA synthesis defines the minimum component that can be described as RNA polymerase 2) transcription initiation factors: provide specific regulatory control for a transcription unit 3) termination factors Spring

4 24 MULTIPLICATION MATURATION Cap Cap CAPPING SPLICING Addition of polya Cap A n 5 3 TRANSCRIPTION DNA

5 Structure of the Transcription Unit 1) the DNA template contains cis-acting sequences that direct entry of the transcription initiation complex promoters specify where RNA polymerase binds and where transcription initiates; they are located near the 5 start site enhancers up-regulate the transcriptional activity of a given promoter and act at a distance in a direction-independent manner 2) the DNA template also contains signals that result in transcription termination Spring

6 Translation Genetic Code: genetic information is stored in the form of nucleotide triplets; each triplet is termed a codon and it codes for 1 amino acid What are the components required for translation? mrna (template) trnas: small RNAs of ~75-80 nucleotides;» each trna recognizes only one amino acid to which it can bind» amino acyl-trna synthetase is responsible for charging trnas with amino acids ribosomes» large complexes of protein and RNA that provide a structural and perhaps enzymatic framework for protein synthesis to occur» allow the positioning of the mrna codon and trna anticodons to come together» ~30% of the dry cell mass is from ribosomes» ~20,000 per cell» polyribosome or polysome - a mrna being translated simultaneously by several ribosomes Translation has three steps: 1) Initiation: involves the reactions that precede formation of the peptide bond between the first 2 amino acids of the protein 2) Elongation: includes all the reactions from synthesis of the 1st peptide bond to addition of the last amino acid 3) Termination: encompasses the steps that are needed to release the completed polypeptide chain Spring

7 Protein Targeting Proteins destined for specific locations within the cell (nucleus, golgi, plasma membrane) contain specific targeting signals. When? post-translational transfer» nuclear proteins» mitochondrial proteins co-translational transfer» proteins bound for the ER and golgi How? signals on the protein receptors on target organelles that recognize specific signals Rough ER - membrane bound polysomes Smooth ER - lack polysomes Spring

8 Molecular Techniques Spring

9 Molecular Techniques Problem: The needle in the haystack problem : How to find a single gene in a complex population of DNA 50µg of DNA displayed on a gel and you can find a single 1kb segment (or about 17 picograms of DNA) Solution: Probe the DNA with a complementary nucleic acid probe and let the probe anneal to its target DNA 1. Blotting: Definition: The process of detecting a target molecule (DNA, RNA, or protein) by probing a solid support which has immobilized on it a complex population containing the target molecule Usually the target molecule represents a minor component of the population (<0.1%) Requires both a target and an appropriate probe/detection system 2. Types of blots Southern: target molecule is DNA Northern: target molecule is RNA Western: target molecule is protein South-Western: DNA-protein North-Western: RNA-protein Spring

10 3. Types of Probes Southern and Northern: DNA or RNA probes Western: antibody probes 4. Detection Systems Southern or Northern: 32 P or Chemiluminuses Western: 125 I, or enzymatic (light, precipitant) Southerns 1. Uses - Physical map of the gene - Determine the number of copies per genome - Information about similarities to other genes (multigene families) and organisms (zoo blots, garden blots) 2. Experimental approach A. Display target DNA - Isolate DNA - Digest DNA with appropriate restriction endonuclease(s) - Run Digested DNA on agarose gel - Denature DNA in high NaOH, high NaCl - Transfer DNA to immobilized support (nylon or nitrocellulose) - Fix DNA to membrane (UV, NaOH, baking) Spring

11 Southerns cont d B. Generate Probe - 32 P dntps and nick translation - Denature probe C. Hybridization - Pre-hybridize with unlabeled non-specific DNA» Block remaining unoccupied sites - Hybridize with labeled probe» (probe will base pair with complementary sequence)» - Hybridization conditions controlled by temperature and salt concentrations - Wash away unbound probe» stringency : a measure of the harshness of the washes. - Expose blot to film D. Interpret results Spring

12 Northern Blotting 1. Uses - Indentifies size of mrna - Quantitates level of transcription of a particular message - provides info on the number of distinct trancripts a gene may produce - is used to examine tissue specific and temperal expression of a gene 2. Experimental approach A. Similar approach as Southerns except RNA is uncleaved and run on denaturing gel systems B. May select poly A RNA or total RNA C. Must be careful to select appropriate complementarity for probe. Spring

13 Western Blotting 1. Uses - similar to those using Northern analysis - provides info on the level of protein expression - can identify protein molecules that may be related based on their antibody reactivity - can be used to examine tissue specific expression of a protein 2. Experimental approach A. Display protein population on SDS-PAGE B. Electroblot to nitrocellulose C. Pre-Hybridize with junk protein D. Hybridize with primary antibody (target specific antibody) E. Wash to remove unbound primary antibody F. Incubate with secondary antibody linked to enzyme tag binds to primary antibody (Goat anti-rabbit) G. Wash H. Add color substrate for enzyme. Spring

14 Western Blotting Spring

15 Antigen Detection & IF Spring

16 Immunoprecipitation Spring

17 Molecular Probes What are probes? Molecular tags - we usually use specific molecules that are labeled to use as probes DNA/RNA probes 32 P 1) End-label: sometimes used for sequencing, provides low specific activity 2) Uniformly labeled:a) nick translation b) random primed c) ribo probes Nonradiactive labeling systems Antibody probes : a) label 2 antibody with 125 I b) use enzyme-linked 2 antibody Spring

18 Libraries 1. Definition: A collection of independent clones that are derived from nucleic acid fragments produced from a specific organism or tissue The construction of a library is an attempt to represent (clone) the entire DNA or RNA population of an organism or a particular cell type. Ex: Human DNA library Ex: Developing chick embryo cdna library Shotgun approach: clone all sequences 2. Types of libraries Genomic library: the cloning of fragments which represent the entire DNA or RNA genome of the organism. cdna library: the cloning of the RNA (usually mrna) population of an organism or particular cell type 3. Why make libraries? Essential for isolating genes Understanding genome complexity Understanding cellular differentiation Construction of full-length cdna virus genomes or collection of virus genes Spring

19 Restriction Enzymes Nucleases - enzymes that degrade DNA (or RNA). Some are very specific where they cleave ( cut, digest ), some are not. There are two types: Exonucleases - digest DNA starting from the termini ( chew- back ). Circular and supercoiled DNAs are resistant. Endonucleases - digest DNA internally. Three groups have been characterized. Types I and III carry a modification (methylation) and an ATP-requiring cleavage activity in the same protein. Type I enzymes cleave randomly whereas Type III cleave DNA at specific sites. Type II enzymes do not carry the modification activity on the same protein, do not require ATP, and they cleave at specific sites. Type II enzymes generally recognize a sequence element of 4, 5, or 6 nucleotides in length with two-fold symmetry (dyad symmetry). What is the natural function of restriction endonucleases and how were they initially identified? Host-controlled restriction / modification systems Bacteriophages: viruses that infect bacteria. They enter through specific receptor molecules, replicate at the expense of the bacterial host, and usually lyse the cell and initiate another round of infection. Phage usually exhibit host specificity. Spring

20 Sequencing Background 1. Cloning of genes alone is useless What you want to know is how the gene functions (function of the encoded polypeptide) 2. Since the early days of gene cloning (1970s), methods were developed to determine the primary sequence of cloned DNA (inefficient, error prone methods) 3. General principles: separation based on size 4. Requirements for modern sequencing a. Pure source of DNA (easy with cloned DNA) b. Sequence only one strand at a time c. Generate sets of radiolabeled single strands containing all possible chain lengths from 1-n 5. Two DNA sequencing methods: a. Chemical sequencing (Maxam-Gilbert) b. Enzymatic sequencing (Sanger) Spring

21 Mutagenesis What is a mutation? An alteration of the primary sequence of genetic material What is a mutant? A strain carrying one or more mutations One may speak about the mapping of a mutation, but one cannot map a mutant. Likewise, a mutant has no genetic locus, only a phenotype. Different types of mutations: 1) Insertions - single or multiple nucleotides 2) Deletions - single or multiple nucleotides 3) Substitutions - a change of the nucleotide 4) Revertants - a. same site b. second site What is mutagenesis? The deliberate alteration of the genetic material Why? 1) Map regions of interest 2) Study regulatory sequences 3) Study protein function 4) Identify regions of molecules which interact with one another 5) Create new activities 6) Correct defects in normal genes Two general approaches: Random and Site-specific Spring

22 Polymerase Chain Reaction I. The power of PCR Advantage: the detection and specific amplification of a single nucleic acid molecule in a complex population Ex: a single grain of sand in a swimming pool full of sand Ex: a single gene or mrna in a single cell ll. PCR Procedure 1. Doubling effect 2. Repeated cycles of three steps for basic PCR: i) Denaturation - heat denature target DNA to form single stranded DNA ii) Annealing - anneal flanking primers by reducing temp iii) Extension - extend DNA strands using polymerase 3. PCR of RNA: requires additional step first strand cdna is synthesized from the RNA templat using reverse transcriptase 4. Theoretical amplification is 2 n, where n is the number of cycles Starting with 1 molecule and 30 cycles = 10 9 molecules Practical amplification Variables affecting PCR i) primers- strength of complementarity ii) length of target - under 2kb easy, 30 kb limit(?) iii) DNA polymerase - true thermal stability and fidelity iv) Kinetics- limiting primers, dntps 6. Problems i) contamination ii) errors Spring

23 PCR cont d Spring

24 PCR Mutagenesis Spring