MBG 2040 Review.pdf

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University of Guelph
Molecular Biology and Genetics
MBG 2040
Mark Baker

MBG 2040 Review Package 5’ 4’ 1’ A nucleotide, with the Forms covalent carbons labelled. phosphodiester bond between adjacent 3’ 2’ The 2’ carbon is attached to an H nucleotides only in DNA, i.e. the sugar is 2- Strong acid group: represents major charge (-) at biological deoxyribose, and is attached to a hydroxyl (OH) group in RNA, i.e. (neutral) pH ranges the sugar is ribose, both are pentose sugars Nucleic acids composed of repeating subunits called nucleotides Pyrimidines: One ring -> Uracil, Cytosine, Thymine Purines: Two rings -> Adenine, Guanine 1 In this diagram there should be a free phosphate group on the 5’ end (ie the top of this diagram), and there should be a free OH group on the 3’ end of this molecule (ie on the bottom of this diagram) Adjacent nucleotides are joined by phophdiester bonds that join 3’OH of one sugar to 5’OH of adjacent sugar giving DNA chain polarity DNA structure ideal for storing genetic information because of the strong covalent bonds and the weaker hydrogen bonds (although many), and hydrophobic bonds (association of nonpolar groups with each other) stacked base pairs provides hydrophobic core Supercoiling controlled by topoisomerase (make single or double stranded breaks in DNA then seal them controlling the degree of DNA coiling, organized collapse into folds aided by RNA and proteins Single human cell contains ~2m of DNA in its nucleus Aids in chromosome packaging, DNA is constrained contains store of free energy wants to unwind, released energy used in opening DNA including replication and transcription and repair Famous Scientists and their contributions (good chance of a matching question with some or all of these): Griffith  “The transforming principle” used streptococcus colonies to prove that DNase is the genetic material, when treated with protease and RNase Avery, MacLeod, and McCarthy  proof that the “transforming principle” is DNA Alfred Hershey and Martha Chase (sometimes just Hershey and Chase)  genes are made of DNA… DNA was taken up by the bacterial cells, not the protein of the viral coat. Maurice Wilkins, Francis Crick, James Watson, Rosalind Franklin  The DNA double helix structure. Note Rosalind Franklin = X-ray diffraction photo of DNA; Watson and Crick =model building and inference skills; double helix features complementary base pairs, anit-parallel strands, right handed double helix (B-DNA), 10 base pairs per turn Erwin Chargaff  Data on DNA base composition, see Chargaff’s rules in lecture slides. Used paper chromatography to analyze nucleotide composition of DNA, in all organisms A=T, G=C, purines= pyrimidines (A+G=C+T) and A+T/G+C can vary between organisms, led to recognition of base pairing, opposite polarity of the 2 strands, A-T has 2 H-bonds, G-C has 3 H- bonds 2 Meselson and Stahl  determined that the semi-conservative model for DNA replication was indeed what happens. They used DNA containing heavy nitrogen ( N) and light nitrogen ( N) 14 to prove their ideas. Q using these ideas in Tutorial 5 Maxam and Gilbert  first method of DNA sequencing (no longer used) Sanger  Most widely used current method for DNA sequencing (more on this further into the package) K. Mullis  Developed PCR (Polymerase Chain Reaction) Barbara McClintock  work on chromosome breakage in 1941 (broken chromosome ends fuse, but normal ones don’t) Chromatin isolated made mostly of DNA and basic (+) histones, heterogeneous, largely acidic group of non-histone proteins also associated with chromatin although there is amount and composition varies depending on how DNA is isolated, small amount of RNA is present Major Histone types: H1, H2a, H2b, H3 and H4, highly conserved proteins Histones are the organisational proteins that our DNA is coiled around, they contain an abundance of Arginine and Lysine residues (positively charged at pH=7). Since DNA is largely negatively charged (think of all the phosphate groups!) the positive charges allow the histones to interact easily with the DNA. 2-nm double-stranded DNA molecule. 11-nm nucleosomes. 30 nm chromatin fiber. Organization around a central scaffold. (Note that the measurement in this figure for the DNA molecule should be 2nm, not 20nm). These measurements are important and could come up on the exam (they have in the past) as well as the measurements between bases (0.34nm) and the measurement for one complete turn of the double helix (3.4nm). 146 nucleotide pairs of DNA wrapped as 1 ¾ turns around an octamer of histones with linker DNA varying in length from 8-114 nucleotide pairs Metaphase chromosomes- 2 identical sister chromatids Centromeres- directs movement of chromosome into daughter cells every time cell divides, mammalian centromeres contain satellite DNA sequences (AT rich) repeated thousands of times, proteins assemble on centromere to form kinetochore (attachment site for spindle fibers that pull chromatids apart in cell divisions 3 A complex structure protects Telomere structure: chromosome ends from degradation - first suggested by Barbara McClintock’s work on chromosome breakage in 1941 (broken chromosome ends fuse, but normal ones don’t) Protects ends of chromosomes and genetic material from degradation and fusion to other chromosomes However, the repeated sequence would be something I could see a multiple choice Q about. Or a short answer question asking how this protects the ends of the chromos.mes Localizing specific complementary sequences in chromosomes by “in situ” hybridization: squash cells on slide, denature DNA, incubate with labelled probe, visualize by microscopy Localizing centromeres and telomeres in human chromosomes- exploits in situ hybridization and fluorescently-labeled centromeric or telomeric DNA probe fragments DNA replication in bacteria- bidirectional beginning at replication origin oriC (245 bp)- AT rich region that undergoes strand separation (dNMP) + nNTP  (dNMP) n+1 + PP i Deoxyribonucleic chain + triphosphate chain extended by 1 + 2 inorganic phosphates Basic requirements of DNA Replication - Primer DNA with free 3'-OH - Template DNA to specify the sequence of the new strand - Substrates: dNTPs (deoxyribonucleic triphosphates) - Mg2+ - maintains conformation of DNA polymerase - DNA polymerase 4 5 DNA polymerases in E.coli, I and III are used for chromosomal DNA replication, II, IV, and V are used for DNA repair functions. Pol. I -aids in removal of RNA primers on lagging Pol. III-main replicative polymerase; highly strand processive stays on for long time -has 5’ to 3’ polymerase activity to copy DNA -has 5’ to 3’ polymerase (incorporating one nucleotide at a time -lacks 5’ to 3’ exonuclease activity after removal of primer) and 5’ to 3’ exonuclease (can’t digest DNA ahead of it) (allows to chew away in 5-3 for removal of primer) activity -Proofreading: has 3’ to 5’ exonuclease activity Proofreading: has 3’ to 5’ exo activity - has catalytic core that could easily fall off held on by 2 -not highly processive; short tract synthesis, doesn’t stay β subunit monomers that create a sliding ring to hold on DNA for long time, works on lagging strand polymerase to DNA molecule All DNA polymerases require a primer strand which is extended and template strand which is copied Polymerization- have absolute requirement for 3’-OH on primer strand and all DNA synthesis occurs in 5’ to 3’ direction (polymerase activity) Editing function 3’ 5’ exonuclease activities of DNA polymerases proofread nascent strands as they are synthesized removing any mispaired nucleotides at the 3’ termini of primer strands Polymerases other than I and III you will not be asked details, just know that they exist. I would know the differences between I and III, and relate them to where they would fit in a DNA replication scenario (i.e. be able to draw them on a replication fork or bubble diagram). Discontinuous DNA replication on lagging strand creating okazaki fragments Replication mechanism requires: - Topoisomerase- relieves tension makes nicks in the strands - Helicase- only on 5’-3’ strand, catalyzes the unwinding of the parental double helix for polymerase, opens duplex for copying - Single-strand DNA binding protein (SSB)- protects and keeps DNA straight, knocked off by polymerase, without would form hair pin structures - primase – forms short track of RNA primer for initiation, on 3’ strand - DNA polymerase III – removes primer by hand off and switches with poly I to remove primer - DNA polymerase I - DNA ligase Okazaki fragments -short lagging strand DNA fragments 5 rNMP- degraded ribonucleic monophophates How is DNA replication different in eukaryotes vs. prokaryotes? - Multiple origins of replication, bi-directional replication from each origin - Occurs only during S-phase - Nucleosomes (disassembled and assembled as DNA is replicated) - Telomeres (review telomere problem in notes) - Shorter RNA primers and Okazaki fragments Disassembly and assembly of nucleosomes is tightly coupled and rapid during DNA synthesis Telomerase- resolves terminal primer problem (DNA polymerse can’t replicate terminal DNA segment of lagging strand (no 3’OH)- extends 3’ end of parental strand, prevents the ends of chromosomes from becoming shorter, human telomerase sequence: 3’AAUCCCAAU5’ DNA polymerase binds to RNA primer left by telomerase and fills in to the last fragment Most human somatic cells lack telomerase activity, shorter telomeres associated with cellular senescence and death, diseases causing premature aging associated with short telomeres (progeria, Werner’s syndrome) These types of things are important and you will see that differences between prokaryotic pathways and eukaryotic pathways is a recurring theme in this course… this should be a hint that there might be a question about some of these differences somewhere along the way. DNA Sequencing: Sanger sequencing-uses terminating nucleotide analogs of A, T, G and C called 2’3’dideoxynucleotide triphosphates (ddTTP, ddATP, ddGTP, ddCTP), ddNTP contain no 3’OH therefore DNA chain can’t be extended by polymerase and synthesis stops when ddNTP incorporated Set up 4 tubes - Reaction solution with: - - Mg2+ - - Template DNA strand - - Primer strand (3’ OH group) - - DNA polymerase - - 4 dNTP’s - **each tube gets ONE of ddA, ddC, ddG, ddT (this is the most important!) - ** dNTP/ddNTP ratio is ~100:1 - - 6 * chain terminating ddNTPs are inserted opposite their complementary template bases. because of the above ratio, a fraction of fragments in each tube will be end- labelled with a ddNTP, thereby indicating the complementary nucleotide on the template strand. The ddNTP’s are each labelled with a different colour fluorescent label. When the truncated DNA molecules are passed through the detector (in order of size, smallest first) they are read out as colour signals. A sequencing read is then output by a computer and is sent to the scientist. As you can see in (B) in the figure the read is not perfect, under some of the peaks there is a smaller peak of a different colour. This is normal as some of the time the wrong base might be incorporated but the peak represents the majority of the DNA molecules of that size have that particular label. Note: The beginning (very small DNA molecules) and end (very large DNA molecules) are very hard for the detector to determine so the outputs are a lot more “messy” around these regions. Usually this is ok because experiments can be designed to leave space around the region of interest, or multiple sequencing runs can be done that overlap significantly so that you are sure of these “messy” areas. Polymerase Chain Reaction (in vitro): N 2 amplification, where N is the cycle number. Example: if you start with one DNA molecule, after 5 cycles you will have 2 DNA molecules (32 DNA molecules) You should be able to describe the journey of a single DNA molecule through a few rounds of PCR (drawing the results after 1,2, and 3 cycles). You should also be able to answer questions that give you a starting number of DNA molecules and ask for the amount you will have after a certain number of cycles. Or that give you starting and ending amounts and ask for the number of PCR cycles that have happened. Protein Expression in Bacteria (keep in mind for this next section differences between prok and euk) 7 Transcription and translation are tightly coupled in prokaryotes (as the RNA is being transcribed from the DNA… before it comes off of the DNA there are already ribosomes on it, translating in into protein molecules). mRNA is short-lived in bacteria because of this. This diagram is misleading because it seemslike the processes are more separated than they are in reality. Protein Expression in Eukaryotes Transcription (nucleus) and translation (cytoplasm) are uncoupled (they happen in 2 different locations within the cell) mRNA is processed before leaving the nucleus to increase its longevity (5’ cap and poly-A tail), and to remove introns etc. Know that there are different types of RNA and that not all of them encode protein. Know what each type is for (i.e. tRNA, rRNA etc.) Messenger RNAs (mRNAs)- intermediates that carry genetic information from DNA to ribosomes Transfer RNAs (tRNAs)- adaptors between amino acids and the codons in the mRNA, links mRNA to protein synthesis Ribosomal RNAs (rRNAs)- structural and catalytic components of ribosomes, part of ribosome, part of translation Small nuclear RNAs (snRNAs and snoRNAs)- spliceosomes (remove introns) and rRNA, tRNA modification respectively Micro RNAs (miRNAs, siRNA and RNAi)- short single-stranded RNAs that block expression of complementary mRNAs Many RNAs do NOT encode protein 8 Also called the anti-sense strand and the non-coding strand Template and transcript are anti-parallel Also called the sense strand and the coding strand When transcribing DNA into RNA there are 2 approaches. 1. You can make complimentary base pairs with the Template strand (also the anti-sense strand and the non-coding strand) 2. You can just write down the sequence of the Non- template strand (coding strand or sense strand) and just swap the T’s with U’s Transcription and translation are not coupled in eukaryotes and most transcripts must be transported to the cytoplasm Transcription is a chemical reaction: (rNMP) + rnTP  (rNMP) n+1 + PP i rNTP= A, U, G, C, requires DNA dependent RNA polymerase similar to DNA synthesis except: precursors are ribonucleoside triphosphates (rNTPs, incorporated as monophosphates), only one strand of DNA is used as template, RNA chains can be initiated de novo (no primer required), RNA molecule will be complementary to DNA template (anti-sense) strand and identical (except uridine replaces thymidine) to the DNA non-template (sense) strand, RNA synthesis is catalyzed by RNA polymerases and proceeds in 5’3’ direction 9 RNA polymerase bind, unwinds and joins first 2 nucleotides In prokaryotes the growing RNA chain would be covered in ribosomes already translating it into protein even before it is Complimentary detached from the DNA. nucleotides In eukaryotes the 5’ cap is continue to be added, unwinding added as the chain is growing but the poly-A tail is added after and rewinding the RNA dissociates from the DNA. The introns are also spliced out after the transcription process is done. Transcription stops, signal tells RNA it has reached end of gene, polymerase will leave DNA, translation will start when RNA strand is long enough and ribosome will attach to 5’ end E. coli RNA polymerase: tetrameric core: α , β, β’ (transcribes any DNA), Holoenzyme: α , β, β’ σ 2 2 (transcribes specific genes) sigma gives specificity allows polymerase to start at the beginning without there is no specificity and DNA could transcribe anywhere Functions: α- assembly of the tetrameric core, β- ribonucloside triphosphate binding site, β’- DNA template binding region, σ- initiation of transcription at promoters (binds at -35 (5’TTGACA3’non- template), localized unwinding at -10 (5’TATAAT3’ non- template) start signal) Transcription initiates about 5-9 base pairs down from the end of the -10 sequence, 5’ end of the RNA is usually a purine Transcription bubble- RNA polymerase has helix unwinding (topoisomerase would be working ahead making nicks for unwinding) and rewinding activities confined within polymerase to protect from nuclease and sealed quickly to avoid degradation, locally unwound segment of DNA ~ 17 base pairs Rho-independent or intrinsic termination- DNA template strand has 2 sequences separated by a few bps that are palindromes and are able to base pair and form a hairpin structure followed by UUUU (unstable base pairing) on RNA sequence to aid in chain termination, common in bacteria, weak H-bonding at U:A residues allows mRNA release from DNA when polymerase pauses at terminator 10 Eukaryotic Transcription: Puffs (balbiani rings) in Drosophila polytene salivary chromosomes are sites of localized unwinding due to gene transcription Α-Amanitin is an inhibitor of RNA polymerase II- deadly toxin from a mushroom Plants have 2 more RNA polymerases (IV, V) in nucleus, siRNA for inhibiting certain genes Modifications to Eukaryotic pre-mRNAs - A 7-Methyl guanosine cap is added to the 5’ end of the primary transcript by a 5’-5’ phosphate linkage. Binds proteins and protects 5’end from degradation. - A poly(A) tail (a 20-200 nucleotide polyadenosine tract) is added to the 3’ end of the transcript. The 3’ end is generated by cleavage rather than by termination. Binds proteins and protects 3’end from degradation. - When present, intron sequences are spliced out of the primary transcript, doesn’t always involve every intron, regulation to control type of splicing that occurs causing slight variation - Alternate splicing permits different exons to be combined together to produce different proteins from the same transcript- occurs in an estimated 60-80% of our genes! This means that even though we might only have a certain number of genes that does not mean that we are restricted to only that number of different proteins. These are the consensus sequences that are part of an RNA polymerase promoter in eukaryotes. There are some similarities to the prokaryotic promoter, but you should always keep in mind the differences between the two, especially in this case where there are many more consensus sequences than for prokaryotes. Transcriptional start (+1) is found approximately 20 bases downstream of the TATA box. [NOTE that this is DIFFERENT from the ATG start codon which is for translation] ^Know this sequence- All help direct polymerase to right spot on DNA positions the polymerase for transcription, found in 11ny genes and very important Initiation- promoter sequence elements bind transcription factors in ordered sequence; transcription factors (TFII(transcription factor): D- binds to TATA box, A, B, F-promotes unwinding, E) form pre-initiation complex, core factors to help polymerase transcribe, prepares DNA for unwinding and recruits RNA polymerase and (TFIIE) to form transcription bubble and initiate transcription, RNA polymerase II can move through nucleosomes, in transcription the nucleosomes aren’t removed and polymerase can transcribe through it but the nucleosomes need to be altered (FACT protein: modifies histones to allow polymerase II to transcribe), with all the factors (except E) in place polymerase is encouraged to come, E joins after polymerase There are transcription factors that bind to the promoter sequence in order and form a pre- initiation complex (don’t worry too much about what order they bind in etc. but know what at least a few of them are called in case you need to use an example). Some introns are called ribozymes (RNA enzyme) and they are able to splice themselves out of the RNA transcript without an external enzyme they may also be able to reinsert themselves to another gene evolution. Reaction requires G 3’OH, involves sequential phophodiester bond transfers: 3’OH attacks the phosphate (nucleophilic) to break junction, uracil for recognition, intron will be degraded Introns that are not self- splicing need the help of a “spliceosome” or snRNP complex. Introns are spliced out by snRNA- protein (snRNP) complexes called sliceosomes (cleaves at 5’ then 3’, and the exons are joined), spliced by recognizing a junction, must be removed in correct frame Spliceosome- RNA/protein structure- 5 snRNAs: U1, U2, U4, U5, U6, snRNAs associate with proteins to form snRNPs (small nuclear ribonucleoproteins), alternative slicing leads to different proteins (60-80% of our genes) on one gene have multiple exons and some have poly A tails, transcription could stop after either tail giving different proteins with different functions Reverse Transcription: This is the process that is used to make DNA copies of mRNAs … you end up with a cDNA molecule (this is differentiated from the original DNA sequence because it doesn’t have introns). mRNA with a poly A tail, hybridize with Poly T primer, add reverse transcriptase (copies RNA to DNA), degrade RNA with RNase, synthesize a complementary DNA strand using DNA polymerase (RNA fragment acts as a primer), recover gene Microarray or DNA Chip Technology: spot specific human gene sequences onto well of a plate, prepare mRNA from normal and tumor cells, make cDNA copy of each but labelled with different fluorescent dye, hybridize microarray and scan, expressed genes that are unique to 12 normal cells fluoresce green, unique to tumor cells fluoresce red, common to both normal and tumor fluoresce yellow; a global approach to study transcription in a cell Regulatory RNAs (miRNA, siRNA, RNAi; micro RNA, small inhibitory RNA, RNA inhibition): A form of post transcriptional control of gene expression They work by creating a double stranded region on the mRNA in the cell, this RNA then gets degraded by the cell and this reduces the expression of the protein that the sequence was designed to interfere with. Gene expression knockdown by RNAi-directed mRNA cleavage, removed by RNA induced silencing complex (RISC), can control gene expression in vivo By convention, peptides are always Amino Acids written from N-terminus to C- terminus because they have directionality. The structure of a peptide bond is also very important to know. Polyribosomes=polysomes in eukaryotic cells Ribosomes are the cellular machinery that translates mRNA into peptide chains. Ribosomes are composed of polypeptides (>50) and RNA- most frequently made RNA (3-4) in approximately 50:50. They are divided into a large subunit and a small subunit. Is an RNA machine with key RNA roles including the formation of peptide bonds; prokaryotic: 31 ribosomal proteins to make large subunit (5S rRNA, 23S rRNA, = 50S subunit, 21 ribosomal proteins, 16S rRNA = 30S subunit, both subunits make 20nm 70S ribosome; Eukaryotic ribosome: 49 ribosomal proteins, 5S rRNA, 5.8S rRNA, 28S rRNA = 60S subunit; 33 ribosomal proteins, 18S rRNA = 40S subunit, both make 24nm 80S ribosome Amino-acid activating enzymes (20)- tRNA synthetases Soluble proteins (translation factors) involved in polypeptide chain initiation, elongation and termination
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