Biology Review Genetics PDF

Nucleotide: - 5 carbon deoxyribose sugar - Phosphate group - One of 4 nitrogenous bases Purine: AG (double) Pyrimidine: CT (single nitrogenous base structure) A=T 30% proportion G=C 20% proportion (Add to 100%) Hydrogen bonds between nitrogenous bases DNA stranded - 2 right-handed (cl...

Nucleotide: - 5 carbon deoxyribose sugar - Phosphate group - One of 4 nitrogenous bases Purine: AG (double) Pyrimidine: CT (single nitrogenous base structure) A=T 30% proportion G=C 20% proportion (Add to 100%) Hydrogen bonds between nitrogenous bases DNA stranded - 2 right-handed (clockwise) helix - 1 complete helical turn every 10 nucleotide - Backbone = sugar-phosphate held by phosphodiester bonds (covalent) - 2 chains held together by H-bonds between bases - Base pairing always purine with pyrimidine - 3’ end with OH group - 5’ end with a phosphate group DNA Eukaryotic in the nucleus DNA Prokaryotic in cytoplasm DNA replication: copying 1 DNA molecule into 2 identical molecules (before cell division) Conservative:one new molecule with old Semi-conservation: two hybrid molecules of old and new stands Dispersive: hybrid molecules with each strand being a mixture of the old and new stands DNA Replication: 3 phases = initiation, elongation, termination Initiation: (start) - Begins at origin of replication - Helicase enzyme unwinds DNA breaking hydrogen bondsbetween complimentary base pairs, forms replication bubble with Y’shape fork at each end. - Single stranded binding proteins help stabilize the unwound DNA at top and bottom (prevent from binding H bonds) (guide synthesis of new polynucleotide strands) - Topoisomerase II relieves stain on double helix (at end near Y-shape) Elongation: (builds new DNA strands)  - Primase enzyme lays down RNA primers (short complementary RNA sequences) used by DNA polymerase II (main enzyme that copies/makes DNA), at staring points of RNA and builds new complimentary strands. - DNA polymerase III adds new nucleotides (in 5’ to 3’ direction) to create DNA stand complementary to the parental strand. - DNA always synthesized 5’ to 3’ - “Reads” DNA from 3’ - 5’ Leading Strand: strand uses 3’ to 5’ template strand (parent DNA) and builds towards replication fork Lagging Strand: short segments away from replication forl, discontinuous (Okazaki fragments) Okazaki fragments: DNA fragments synthesized (built) on lagging stand (each has a primer) DNA polymerase I: removes the primers and replaces with DNA, fills the space by extending the neightbouring DNA Ligase: enzyme bonds okazaki fragments together. Joins sugar phosphate (covalent bonds) Helicase: helps unwind parent DNA Primase: synthesizes RNA primer used to generate Okazaki fragments Single-strand-binding proteins: helps stabilize single-stranded regions of DNA when unwinds Topoisomerase II (gyrase): helps relieve strain on the structure of the parent DNA generated from unwinding double helix DNA polymerase I, II, III: group of emzymes with deffering roles… - Adding nucleotides to 3’ end of growing polynucleotide strand - Remove RNA primer and fill gaps between Okazaki fragments - Proofreading newly synthesized DNA DNA ligase: joins the ends of Okazaki fragments in lagging strand synthesis Gene Expression Information encoded in DNA is used to make protein Flow of information: DNA → RNA → Protein One gene/one-polypeptide hypothesis: one gene is transcribed and translated to produce one polypeptide. Each polypeptide has the own gene. However in eukaryotes, one gene (DNA segment) can encode multiple different proteins (alternative splicing) - Some proteins are made of multiple polypeptides/subunits, thus encoded by ultiple genes. - Example: DNA located in nucleus, proteins synthesized by the ribisomes in the cytoplasm How does ribosome synthesize protein if it doesn’t have acces to DNA? - mRNA is made that is complimentary copy of DNA, mRNA leave nucleus, goes to cytoplasm where ribisome decodes to form protein. Central Dogma: flow of information during gene expression from DNA → RNA → protein  - Directs production of protein at ribosome by controlling amino acid sequence Steps for gene expression Transcription: DNA sequence serves as a template for synthesis of RNA in the nucleus Translation: mRNA sequence serves as a template for the synthesis of a protein in the cytoplasm RNA: single stranded polymer of nucleotides (AGCU) Sugar- DNA(deoxyribose) RNA(ribose) Strand structure- DNA(double) RNA(single) mRNA: end product of transcription. Translated by ribosomes into protein (copy of DNA molecule) rRNA: binds with ribosomal protein to make a part of ribosomes tRNA: brings amino acids to ribosome for protein synthesis (translation) Transcription Events: - Initiation - Elongation - Termination More proteins involves in eukaryothic transcription Initiation: - RNA polymerase (enzyme catalyzes synthesis of RNA) binds to DNA segment that isn’t transcribed and opens the double helix with its own helicase. - Binding site for RNA polymerase is a region before coding region of a gene (promoter region) region high in A and T- requires less energy to break bonds - RNA polymerase travels down the promoter region until it reaches series of A’s and T’s (TATA box) - TATA box signals RNA polymerase that it reached the end of promoter region and can begin reading coding strand to make RNA For every gene, one strand of DNA is transcribed Strand transcribed (template/anti-sense strand) Strand not transcribed (coding/sense strand) Elongation: - RNA polymerase complex moves in the 5’-3’ direction to synthesize an mRNA molecule that is complimentary to antisense strand. In mRNA strand, T replaced by U. Termination: specific nucleotide in DNA template signals to stop transcription. When RNA polymerase reaches this signal, it detaches DNA strand and is reused by the cell. The new mRNA strand is released from the transcription, and DNA double helix reforms. mRNA modification in Eukaryotes: In prokaryotes = transcription and translation can occur simultaneously In eukaryotes = mRNA must undergo modifications before it crosses the nuclear membrane to the cytoplasm. Once modified DNA enters cytoplasm, it undergos translation. 3 modifications that convert precursor mRNA(pre-mRNA) to mature mRNA 1. Adding 5’ cap of modified G nucleotides. The cap is recognized by protein synthesis machinery as an attachement site for the ribosomes. 2. Adding 3’ poly-A tail. It’s a series of A nucleutides that makes mRNA more stable in the cytoplasm - protect the strand from restrictive enzymes 3. Removal of Introns (called splicing) Introns - non-coding regions are removed Exons - coding regions are joined together - This process preformed by snRNA and snRNP proteins, form large spliceosome complex. - Small nuclear RNA (snRNA) in complex with proteins are called small nuclear ribonucleic particles (snRNPs) - These assemble with other proteins to form Spliceosome - snRNA bind to specific mRNA sequences at the beginning and end of an intron forming a loop - The loop is removed and the remaining exons are linked Alternative splicing = allows one gene to code for more than one protein that are specific to that cell. Translation The second stage of gene expression, translates nucleic acid code of mRNA into the amino acid code of a protein. Translation occurs on the ribosome in the cytoplasm or attached to the ER. mRNA: contains genetic information that detewrmines the amino acid sequenxe of a protein tRNA: has a anti-codon that base pairs with a codon on the mRNA and has the corresponding amino acid attached to it, according to the genetic code. Ribosomes: made of rRNA and proteins; involved in protein synthesis Translation factors: proteins that act as accessory factors, needed at each stage of translation tRNA- composed of 3 stem0loops and a single stranded region Anticodon loop: sequence of 3 nucleotides complimentary to an mRNA codon Acceptor stem: single-stranded region at the 3’ end where amino acid is attached mRNA = 5’-3’ Anticodons = 3’-5’ on tRNA Function of enzyme aminoacyl-tRNA synthetase? Enzymes attaching the correct amino acid to a tRNA base on its anticodon (requires energy) - There are 20 different enzymes The genetic code is universal and redundant - A given codon can only code for 1 amino acid  - Many different codons that code for amino acid (UCU, UCC, UCA, UCG all code for serine) 3 stop codons = UAA, UAG, UGA Wobble Hypothesis: 3rd nucleotide in an anticodon is less important than first 2 (binds weak). 3rd nucleotide is in ‘wobble’ position Ribosomes: complex of proteins combined with rRNA 2 subunits - small and large One binding site for mRNA Polyribosome: complex of multiple ribosomes. Attached to and translate single mRNA at same time 3 binding sites… 1. P site: peptide, holds one aa - tRNA and the growing chain of aa 2. A site: (acceptor site, accepts incoming tRNA) amino, recieves tRNA with the next aa to be added to chain 3. E site: exit, releases the used tRNA back into the cytoplasm Translation phases 1. Initiation 2. Elongation 3. Termination Initiation: components of translation (mRNA, tRNA, ribosomes) assemble - Small ribosomal subunit binds to mRNA; initiator tRNA with anticodon UAC pairs with mRNA start codon AUG - tRNA carries methionine - Large ribosome subunit completes the ribosome. Initiator tRNA occupies the P site. A site is ready for next tRNA Elongation: - Code Recognition - Peptide bond formation (between adjacent amino acids) - Translocation and release of uncharge tRNA (ribosome shifts over, empty tRNA leaves from E sits, repeats) Termination: Release factor binds to stop codon + entire assembly comes apart (protein is released) Mutations Mutation: change in nucleotide sequence of organisms DNA or RNA of a virus - Mistake in DNA replication, or environmental factors - Mutations in reproductive cells, mutations in DNA of single celled organism affects next generation - Mutations in body (somatic) cells affect daughter cells but not future generations Single-gene mutations: change single DNA nucleotide Chromosome mutation: changes in chromosome structure Point mutations: single gene mutations from change in single base pair with a DNA sequence 3 types of point mutations… 1. Substitution: one base for another 2. Insertion/deletion: one or more bases 3. Inversion: two adjoining base pairs 4 groups of mutations… Mutations from substitution point mutation: 1. Silent mutation- no effect on amino acid from codon because of redundency in genetic code. Happens on 3rd base in codon because base binds less specifically than first 2 (wobble hypothesis) 2. Non-sense- changes amino acid into stop codon. Results in incomplete protein structure (protein will be shorter - nonsense) 3. Missense- still codes for amino acid but not correct aa. Protein structure will have different amino acids. Changes expression - function of protein 4. Frameshift: one or more nucleotides inserted or deleted from DNA sequence. Causing reading frame of codones to shift one - multiple missense/nonsense effects. (insert/delete of 3 nucleotides won’t cause frameshift) Conservative: 1 amno acid replaces by new similar amino acid (similar function and shape) Non-conservative: 1 amino acid replaced by very defferent one (different function and shape) Transition: replaces purine + purine or pyrimidine + pyrimidine (A with G or T with C) Tranversion: replaces puring + pyrimidine or pyrimiding + purine (A with T or C) Chromosome mutations: changes in chromosomes and genes 3 main types are… 1. Deletion or duplication of portions of chromosomes 2. Inversions - chromosome segment broken and re-inserted in the opposite direction 3. Translocations - section of one chromosome broken and fused to another Spontaneous mutation causes - Normal molecular interactions - Incorrect base-pairing by DNA Polymerase (replication) - Transposition, specific DNA sequences (called transposons or jumping genes) move within and between chromosomes Induced mutation causes - Physical mutations (change structure of DNA, 2 adjacent T covalently linked together) - Chemical mutations (enter nucleus and react with DNA) Regulation of gene expression in prokaryotes Gene regulation: control of the level of gene expression (active or not), how its active, amount of protein available Constitutive gene: always active at constant levels because essential for cell survival  - Most genes are regulated so only expressed when needed Regulation of gene expression in prokaryotes 3 levels: 1. Transcription 2. Translation 3. After a protein is synthesized Most common regulation is during initiation of transcription In Prokaryotes- related genes cluster in region under control of single promoter (where RNA polymerase binds) Region containing these components is an operon Lac operon encode enzymes that break down sugar lactose Regulatory region contains… 1. Promoter: where RNA polymerase binds, involved in transcription of lactose metabolizing enzymes 2. Operator: DNA sequence a protein(repressor) binds to inhibit transcription initiation 3. CAP binding site: DNA sequence which CAP (catabolic activator protein) binds to increase the rate of transcription The control region responds to the presence or absence of glucose and lactose When lactose present = allolactose produced Activating the lac operon Glucose Not present lactose not present = transcription off Glucose present lactose not present = transcription off Glucose Not present lactose present = transcription on (FAST) Glucose present lactose present = transcription on (SLOW) Trp (tryptophan) Operon = normally active until repressor turns it off No tryptophan = operon on Enzymes synthesized to make trp Trp present = operon off Try is a co-represson Regulation of gene expression in eukaryotes (both in nucleus and cytoplasm) - Pre-transcriptional (DNA tightly packed, cannot be transcribed, epigenetic modification, DNA methylation, histone accetylation) - Transcriptional (either enhance transcription, repressor molecules to repress transcription) - Post-transcriptional (different mRNA splicing, transport to cytoplasm, mRNA degradation) - Translational (molecules RNA or regulatory proteins can attach to mRNA and prevent translation)  - Post-translational (acitivation and deactivation of proteins, (ex. adding phosphate group), protein degradation) Recombinant DNA: DNA combined from 2 different sources Why do scientists create recombinant DNA? 1. Produce many copies of particular gene 2. Allows a cell to produce a protein is normally doesn’t, can be used by humans (ex have bacterial cells produce human insulin) 3. Allows organisms to have certain characteristics (ex. Pest resistant crops) Restriction endonucleases (restriction enzymes): recognize specific nucleotide sequence (target sequence) within DNA. Cuts DNA strans at specific 4-8 base pair site (restrictive site). Restrictive fragments: cuts that forms identical sets of DNA fragments Staggered ends: most endonucleases produce this, leaves regions called sticky ends at end of fragments. Blunt ends: allows and 2 DNA fragments to combine. When restrictive enzyme comes across restrictive site, it breaks the phosphodiester bonds in hydrolysis reaction. Why are sticky ends more useful? Blunt Ends: don’t have any unpaired bases Sticky Ends: have unpaired bases. Can hydrogen bond with complimentary sticky ends of other fragments. Origin of restrictive enzymes = bacteria Naming restrictive enzymes 1st letter = genus name 2 and 3 = species name 4 = strain Numerals = order of discovery of enzyme from that strain of bacteria How do prokaryote cells protect their own DNA from being digestive from restrictive enzymes? Methylases modify recognition site of respective RE by placing methyl group on one of the bases, preventing the RE from cutting the DNA into fragments How is recombinant DNA made? 1. 2 different fragments cut with same enzyme to produce complimentary single sticky ends 2. Base pairing between complimentary sticky ends brings molecules together 3. DNA ligase covalently joins the strands to produce double-stranded recombinant DNA Ligase: enzymes that creates bonds between restriction fragments (phosphodiester bonds) between 3’-5’ ends (Hydrogen bonds aren’t permanent enough for replication)  - Circular piece of DNA, Plasmid is removed from bacteria cell and cut open using same restriction enzyme. - Cut out human gene is mixed with bacterial plasmid - Sticky ends - Ligase sticks them together Plasmids: small circular piece of DNA that are separate from bacterial chromosome and replicates independently - Plasmids have genes that produce antibiotic resistant proteins How do you know if bacterial cell took up plasmid? Will have gene for antibiotic resistance the plasmid contained How do you know if it actually contained added gene? Recombinant DNA - active lacZ gene or not and contains plasmid only Bacterial Transformation: uses DNA recombinant tech and tools to clone DNA in host cell (accepts foreign DNA) Host cell: accepts DNA (replicates itself and attaches DNA) Vector: carries foreign DNA, vehicle used