Study Guide > University of California, San Diego - BENG 168beng 168 final notes. Chapter 2 – DNA, RNA, and Protein Synthesis.

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Chapter 2 – DNA, RNA, and Protein Synthesis • DNA structure o Composed of nucleotides – deoxyribose + phosphate + base o Bases – adenine (purine), guanine (purine), cytosine (pyrimidine), t... hymine (pyrimidine)  A-T and G-C pairing  2 H bonds for A-T pair, 3 H bonds for G-C pair o Double-stranded helix (discovered by Watson + Crick using X-ray diffraction) • Replication o Each strand of existing DNA molecule is used as a template for producing a new strand o Sequence of growing strand is determined by base complementarity o Goes from 5’ to 3’ end on growing strand, new bases have to link up to 3’ OH on template strand o DNA polymerase binds nucleotides -> forms phosphodiester linkage o Prokaryotes vs eukaryotes:  Prokaryotes have 1 ORI, eukaryotes have multiple ORI  For eukaryotes, replication occurs in multiple sites simultaneously -> ends have to ligated together • Need telomerase to complete telomeres at ends of chromosomes • RNA, protein o Proteins – essential polymers, involved in almost all biological functions, consist of a specific sequence of amino acids  Fold into specific conformations depending on locations of specific amino acid residues + overall AA composition  Properties are determined by properties of side chains  AA linked together by Peptidyl transferase o RNA – intermediate molecules for decoding genetic material, transcribed from DNA  Have ribose instead of deoxyribose, uracil instead of thymine, only 1 stranded  Types: mRNA, tRNA, rRNA • In prokaryotes, all transcribed by the same polymerase • In eukaryotes, each transcribed by different polymerases  RNA polymerase links ribonucleotides together • Binds to promoter sequence to initiate transcription • Euk: alternative splicing gives rise to multiple proteins from same gene • Terminator sequence signals RNA poly to stop RNA synthesis  Translation: • Prok: occurs simultaneously w/ transcription • Euk: occurs separately from transcription (transc in nucleus, transl in cytoplasm) • Primary transcripts in euk are modified w/ polyA tail and 5’ cap first • Reading of stop codon terminates translation -> termination factor recognizes stop codon and binds to ribosome    Transcription regulation (bacteria) • Operon arrangement: contiguous bacteria structural genes that encode proteins required for several steps in a single metabolic pathway • Operator region: regulatory region that control on/off states of various operons, play essential role in determining if operon is transcribed • Effectors can bind to repressor proteins -> change conformation -> prevent it from binding to operator -> ”turn on” • Alternative: corepressor can bind to inactive repressor -> alter conformation to make it active -> turn off transcription  Transcription regulation (eukaryotes) • Basal set of structural genes maintain routine cellular functions • Transcription mediated by proteins collectively classified as “transcription factors”, bind directly to DNA sequences • Promoters: TATA box, CCAAT sequence, GC box Chapter 3 – Recombinant DNA technology • Restriction endonucleases o Type II – restriction enzymes, bind to recognition sites n DNA and splice at these areas  Can produce sticky ends (overhangs) or blunt ends  IIS subcategory – cut a fixed number of nt away from the recognition site • Useful in preventing reopening of splice sites (nicks)  Methylation of cytosine residues in host DNA prevents Res from cutting at sites  Use ligase to catalyze formation of phosphodiester bonds at nicks in backbone • Plasmid cloning vectors o Plasmids – self-replicating, ds, circular DNA; maintained in bacteria as independent chromosomes  Can be high-copy number or low copy number  Plasmids from different incompatibility groups can be maintained in same cell  Have the basic attributes to serve as vectors for carrying cloned DNA  Have to be engineered first: • Insert unique RE sites for insertion of cloned DNA • Need selectable markers for identifying transformed cells o pBR322  resistance genes for Amp and Tet  BamHI, HindIII, SaII, PstI, EcoRI RE sites  E. coli ORI that functions only in E. coli, is maintained at a high copy number, cannot be readily transferred to other bacteria  Process of cloning/transformation: • Insert GOI into Tet gene using RE site of choice o Final cells lack Tet resistance b/c gene is nonfunctional • Transform plasmid DNA into cells and plate on medium with Amp o Kills off cells w/o plasmid • Use replica plate on medium with Tet and identify colonies that die o Pick those colonies from original and culture o pUC19  Amp gene, lacZ gene, lac promoter, lacI gene, MCS site w/ many RE sites  Cells w/o cloned plasmid should be able to hydrolyze X-Gal to blue color  Nontransformed cells again die in presence of Amp  Successfully transformed cells can’t hydrolyze X-Gal: lacZ gene is nonfunctional due to transformation disrupting codon sequence • Creating and screening a library o Treat a DNA sequence with RE and carry out partial digestions (low RE [] or short incubation time) o Clone the sequences into plasmid vectors o Identify clones w/ target sequences: DNA hybridization, immunological screening, assaying for protein activity, functional complementation • Cloning sequences encoding eukaryotic proteins: o Use reverse transcription to create cDNA for cloning (reverse transcriptase enzyme) o Secondary strands created from RNA-cDNA using ribonuclease to nick mRNA -> provide free 3’ OH group for DNA synthesis, 5’ activity of DNA polymerase I removes ribonucleotides that are encountered o Screen using DNA hybridization • Transformation methods o Electroporation – “electric-field mediated membrane permeabilization” o Conjugation – effective contact between donor cell and recipient cell, plasmid genes encoding conjugative functions Chapter 4 – Chemical Synthesis, Amplification, and Sequencing of DNA • Chemical synthesis of DNA o Phosphoramidite method  1) Linking first nucleotide to column -> 2) washing -> detritylation -> 3) washing -> 4) activation + coupling -> 5) washing -> 6) capping -> 7) oxidation  -> n cycles of step 2 to step 7 -> step 2) -> remove oligo from colum -> purify o Uses of synthesized oligonucleotides  Use as probes to screen genomic libraries  Use for cloning DNA, creating unique cloning sites (linkers containing RE sites, adaptors for creating cloning sites in vectors)  Key components for assembling synthetic genes • Uses: large scale protein production, testing protein function after altering codons, creating sequences w/ novel properties  Creating full size genes • 1) Create set of overlapping, complementary nucleotides • 2) synthesize specified set of overlapping oligo, ~60 nt long, overlaps ~20 nt long   • Polymerase Chain Reaction (PCR) o Amplification procedure for generating large quantities of DNA in vitro o Components: primers, template sequence, thermostable DNA polymerase, dNTPs o Steps  Denaturation: 95 C, denature the template DNA  Renaturation: 55 C, primers anneal to template strand  Synthesis: 75 C, dNTPs start annealing to template strand using primers as starting point o Short copies eventually accumulate and outnumber longer copies o PCR amplification of full-length cDNAs  Use oligonuc with oligo(dT) and added sequence (oligo(dT)-primer) + reverse transcriptase to create first cDNA strand  Terminal transferase activity adds mostly dCs to end of each full-length first-strand cDNA molecule  dG primer base pairs with dC tail, acts as template for extending first strand  repeat cycles of forward and reverse primers o Gene synthesis by PCR  Faster, more economical than using overlapping oligonucleotides w/ DNA polymerase + sealing nicks with ligase  Start with 2 overlapping oligonucleotides, recessed 3’ OH groups serve as starting points for elongation -> annealing dNTPs  Add two additional oligonucleotides that overlap with original 2 -> anneal dNTPs  Can synthesize 1000 bp gene in one day • DNA sequencing techniques o Sanger: termination of growing strand using ddNTPs + fluorescence to detect where the strand stops elongating o Primer walking: have primers bind to sequence of interest and sequence the following segment  Must sequence both strands for full accuracy  False priming can lead to erroneous reading, use longer primers to avoid this  Useful if you already have a general idea of the sequence o Pyrosequencing: NGS technique, basis is detection of pyrophosphate released during DNA synthesis  Entails a series of enzymatic reactions, sequence 1 nt at a time  Can be problematic for homopolymer tracts because number of nt is linear with amount of light produced -> can be difficult to gauge number accurately o Ligation: NGS, extend by ligation of short oligos in template dependent fashion  Requires degenerate octamers or nonamers  Each base type gives off a different fluorescence -> can do multiple bases at a time  Perform multiple runs offset from each other by 1 nt   • Large scale sequencing o Shotgun: fragment multiple copies of a DNA sequence with physical force -> end up with overlapping fragments  Clone segments in vivo  Use Sanger sequencing to sequence shorter fragments  Use overlaps to reassemble full genome o Cyclic array (454 technology)  Fragment genome into fragments, anneal to DNA capture beads  Amplify using emPCR (PCR in emulsion bubble microreactors, more efficient)  Place beads into wells and use pyrosequencing to sequence in parallel Chapter 5 – Bioinformatics, Genomics, Proteomics • GFP o Green fluorescent protein, can engineer to respond only in presence of substrate and use for operon/regulatory analysis • Functional genomics o DNA microarray technology  Take cDNA from 2 or more samples, mix and hybridize to an array  Each time has a different fluorescence attached to it  Scan array for emissions and record  Alternatively: screen cRNA synthesized from cDNA  Purposes: • Identify genes whose expression changes in response to a particular biological condition • Identify genes that are coexpressed under different conditions/over a period of time o SAGE (serial analysis of gene expression)  Use recombinant DNA techniques to clone randomly linked short sequences of cDNA to identify expressed genes • Proteomics o Separation and identification of proteins  2D PAGE: gel electrophoresis for separating proteins (by MW and charge)  Use Mass spectrometry to identify proteins after separation • Measure mass/charge ratio, search for matches in protein database o Protein expression profiling:  2d differential in-gel electrophoresis • Similar to PAGE, but compare results from two samples  ICAT method • Use affinity tag and labeled linker (light or heavy labeling) • Abundance of light/heavy ICAT indicates relative abundance of source proteins [Show More]

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