Download Molecular Biology: Transcription, RNA Interference, and Gene Regulation and more Exams Nursing in PDF only on Docsity!
DNA
- Made up of nucleotides (A, T, C, G)
- Structure o Sugar, phosphate, base
- Carries information for genes o Proven by Hersey S Chase experiment – bacteriophage injected genetic material into E. coli
- Synthesis occurs in 5’ 3’ direction
- Chromatin o Complex folding chromosome o Heterochromatin = more condensed, less likely to be transcribed o Euchromatin = less condensed, more likely to be transcribed o Regulating chromatin ▪ Chromatin remodeling complex – distort shape of nucleosome and open up DNA ▪ Histone modification – push/move nucleosomes to open up chromatin
DNA Synthesis
- Process o Happens during S phase of mitosis o Replication origins ▪ Relaxed into euchromatin ▪ Initiator proteins open up DNA helix into replication fork o DNA helicase unwinds double helix o DNA primase binds and generates short RNA sequence (primer) o DNA polymerase binds ▪ Leading strand proceeds ▪ Lagging strand is slower o DNA ligase ligates Okazaki fragments together
- Errors in synthesis o Polymerase may add incorrect nucleotide o Proofreading ▪ Recognizes, removes, replaced o If not recognized mutation
Cell Cycle
- G0 phase o Withdraw from cell cycle – “pause” o Differentiate – branch off and differentiate in function
- S phase
o Synthesis – duplicated chromosomes held together by cohesin rings o Chromatids (identical copies of chromosomes)
▪ Apoptosis (programmed cell death) o Regulated by cell o Neat, contained o Engulfed by neighboring cell o Signaled by caspases/proteases
▪ Necrosis o Any injury that causes harm to cell o Cell broken open inflammatory response
Mendelian Genetics
▪ Test cross – cross between one genotype with homozygous recessive genotype
▪ Mendel’s assumptions o Alleles must segregate from each other o Separate alleles must assort independently o Random fertilization (equal probability of all gametes) o Complete dominance FALSE ▪ Hypomorphic alleles – partial loss of function ▪ Ex. Eye color in flies – can show color gradient (red, orange, apricot, white) ▪ Ex. Flowers – white, red, pink o No lethal alleles or lethal allele combinations FALSE ▪ Loss of function allele – don’t produce protein at all o Each gene affects a diff. trait FALSE ▪ Gene interactions – epistasis
Meiosis
▪ Process o Interphase – occurs after DNA synthesis o Meiosis I o Meiosis II ▪ Bivalent (2) sister chromatids line up next to each other o Synaptonemal complex – protein complex ▪ Lateral elements and central elements
o Chromosomes may break off and cross over – exchange genetic material o Likelihood of recombination events – depends on how far apart they are
Outline of Protocol
- Generate dsDNA encoding H2B
- Ligate dsDNA of H2B to dsDNA encoding GFP generate plasmid
- Generate a large quantity of plasmid
- Check and use restriction alaysis to verify
- Transform human cells with plasmid
- Analyze expression in cells
Polymerase Chain Reaction (PCR)
▪ Process o Heat (denature) o Add primer and hybridize (annealing) o DNA Polymerization ▪ What is required o Template DNA o Two primers o DNA polymerase o dNTPs
Restriction Analysis
▪ Use restriction enzymes to cut plasmid a specific regions ▪ Run it through gel electrophoresis
Sequencing
▪ What is required o ddNTPs – blocks further growth of the DNA molecule – terminates polymerization o Only 1 primer added – only synthesize in one direction o Excess dNTPs ▪ You get multiple copies of every possible combination that terminates at different bases
o Look at linkage
Understanding Gene Function
▪ Generating a knock-out o Alter gene of interest in tissue culture cells o Incorporate cells into germ line eventually give rise to mice in which both copies of target gene are altered ▪ RNAi (refer back to previous) ▪ Ectopic expression o Ex. Drosophila fly – wild type vs. eyeless mutant ▪ Overexpress gene to see effect on phenotype scientists saw eye expressed on leg ▪ Reporter Genes (ex. Drosophila fly) o Take promoter sequences of eyeless and link it to GFP transgene o Eyes then glow with GFP – show where gene is expressed
Transcription
▪ Overview of transcription o DNA unwound by RNA polymerase o Initiation of RNA synthesis ▪ Template strand is read ▪ Coding strand is generated o RNA is elongated until it hits terminator sequence o Newly synthesized RNA is displaced Type of RNA Function mRNA Codes for proteins rRNA Form core of ribosome, catalyze protein synthesis tRNA Adaptors between mRNA and amino acids during protein
- Sigma factor – recognizes promoter sequence where transcription beings
- Binds together
- Sigma factor is displaced when transcription begins o Specific DNA-protein interactions ▪ TATA sequence at 10 nucleotides; similar sequence at 35 nucleotides
- If mutated, will not bind to promoter sequence ▪ High homology across organisms found frequently
o Transcription can occur from both strands in opposite directions ▪ Position/orientation of specific promoter sites determines this o Only one type of polymerase in bacteria ▪ Transcription in Eukaryotes o 3 types of polymerase ▪ RNA pol I – most rRNA genes ▪ RNA pol II – protein-coding genes, miRNA genes, genes for some small RNAs ▪ RNA pol III – tRNA genes, etc. o Eukaryotic promoters ▪ TATA box is core promoter for RNA pol II o General transcription factors ▪ TFIID binds to TATA box
- TBP (TATA-Binding Protein) recognizes it ▪ TFIIB can bind ▪ RNA pol II comes and binds ▪ TFIIH opens up DNA using ATP ▪ Transcription start – RNA pol has a tail that is phosphorylated o RNA pol tail ▪ RNA capping factors – add on G residue that is methylated to 5’ end ▪ Tail of multiple A’s added to 3’ end – polyadenylation factor ▪ Splicing factor
- Splicesosome – protein snRNA complex
- Certain sequences recognized for protein
- Important A “attacks” 5’ splice site – cuts DNA backbone and binds to it
- Free 3’ end jois with start of next exon o mRNA and Nuclear Export Signal
▪ Bound by capping protein, poly-A protein, EJC protein ready for export
- absence of glucose and presence of lactose operon on
o Eukaryotic gene activation occurs at a distance ▪ Because of DNA bends the activator/repressor can interact with transcription complex through mediator ▪ Promoter – region right upstream of general transcription site ▪ Enhancer – region of DNA that transcription factors bind and modify Regulating chromatin ▪ Chromatin remodeling complex – distort shape of nucleosome and open up DNA ▪ Histone modification – push/move nucleosomes to open up chromatin ▪ DNA made more accessible for transcription
Combinational Regulation
▪ Combination of activators and repressors determine gene expression ▪ Ex. Drosophila eve stripe o 4 transcription regulators ▪ 2 activators: Bicoid, Hunchback ▪ 2 repressors: Giant, Kruppel o Remove an activator? See less expression A single transcription factor can regulate multiple genes ▪ Binding site for one factor may be present in promoter regions of multiple genes Combinations of a few transcriptional factors can generate many different cell types ▪ Differentiation
o Anticodon (complimentary sequence) present in tRNA ▪ Ex. UGG is codon, ACC is anticodon ▪ tRNA o Links to specific amino acid via tRNA synthetase ▪ Amino acid is attached to 3’ end by synthetase o tRNA also is 3D and forms helices o Wobble hypothesis ▪ When anticodon binds to codon, the 1st^ bases accurately match ▪ 3 rd^ match can tolerate mismatch ▪ Ribosome o Structure – mix of rRNA and protein ▪ Large and small subunits that complex together o Functions ▪ Bind/move along mRNA ▪ Recognize translation start site ▪ Capture tRNAs ▪ Hold tRNAs in place ▪ Covalently link the amino acids together ▪ Process of translation o Initiation ▪ Ribosomal subunit with translation initiation factors bind to RNA ▪ Initiator tRNA moves along RNA searching for Methionine (start) ▪ Large ribosomal subunits bind (initiator factors dissociate) ▪ Next tRNA is recruited ▪ First peptide bond forms o Elongation ▪ Growing polypeptide chain ▪ tRNA that had Methionine (and following amino acids) are ejected out of exit site o Termination ▪ Hits stop codon instead of loading tRNA there is a release factor ▪ Release factor alters peptidyl transferase activity
- Generates carboxyl end – translation is terminated ▪ Polypeptide chain is released and folded ▪ Regulation of translation o Translator repressor protein ▪ Binds to mRNA at AUG end and prevents translation o Structure of mRNA ▪ Sometimes need to increase temperature to open up mRNA and start translation o Presence of small molecule ▪ Inhibits translation
o RNA interference
