MBG 2040 Final Exam Review Package.docx

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Department
Molecular Biology and Genetics
Course
MBG 2040
Professor
Christine Schisler
Semester
Fall

Description
MBG 2040 Review Package A few notes about this package: - It is NOT a complete study guide, I’m merely giving you guys some review of the key concepts as well as pointing out some things to watch for, common mistakes etc. It is meant to help you in conjunction with your lecture notes and the textbook. This is the type of thing I would present in a review tutorial. - All the figures in these notes are right out of your lecture slides, I may have added text to them or changed them a tiny bit but for the most part they are the same. - Keep in mind that I have not seen your final exam and I do not know what will be on it for certain… these are the concepts I would focus on if I were writing your final… but I’m not - Study lots! This will be a tough exam. Feel free to e-mail me if you have any questions about this package or any other specific questions, but e-mail me early. (E-mailing me the day before your exam might not get you a response in time) PUT “MBG 2040 QUESTION” in the subject of your e-mail (I’ll read it as a priority that way) - Good luck! Pyrimidines: One ring -> Uracil, Cytosine, Thymine Purines: Two rings -> Adenine, Guanine 5’ 4’ 1’ A nucleotide, with - Forms covalent the carbons phosphodiester bond labelled. between adjacent nucleotides 3’ 2’ The 2’ carbon is attached to an H - Strong acid group: represents major charge (-) only in DNA, i.e. the sugar is at biological (neutral) pH deoxyribose, and is attached to a hydroxyl (OH) group in RNA, i.e. ranges the sugar is ribose. 1 2 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) 3 4 Famous Scientists and their contributions (good chance of a matching question with some or all of these): Griffith -> “The transforming principle” 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 Erwin Chargaff -> Data on DNA base composition, see Chargaff’s rules in lecture slides. 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) 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) Knowing the composition of chromatin could make a good multiple choice Q. Histones are the organisational proteins that our DNA is coiled around, they contain an abundance of Arginine and Lysine residues (positively charged). 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 5 well as the measurements between bases (0.34nm) and the measurement for one complete turn of the double helix (3.4nm). Telomere structure: A complex structure protects 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) I would say there is no need to memorize this figure but understand it. 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 chromosomes. Basic requirements of DNA Replication (Know these! And why each of them are important): 6 - Primer DNA with free 3'-OH - Template DNA to specify the sequence of the new strand - Substrates: dNTPs - Mg2+ - DNA polymerase 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 Pol. III-main replicative polymerase; highly -has 5’ to 3’ polymerase and processive 5’ to 3’ exonuclease activity -has 5’ to 3’ polymerase activity Proofreading: has 3’ to 5’ exo -lacks 5’ to 3’ exonuclease activity activity -Proofreading: has 3’ to 5’ exonuclease -not highly processive; short activity tract synthesis 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). 7 Replication mechanism requires: - Topoisomerase - helicase - Single-strand DNA binding protein (SSB) - primase - DNA polymerase III - DNA polymerase I - DNA ligase Okazaki fragments -short lagging strand DNA fragments This is the figure from your notes that is similar to the replication bubble I drew in tutorials (although this is just one fork not the whole bubble). 8 1 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 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: - You should have a basic understanding of Sanger sequencing. (You should be able to write out an explanation of how this method of DNA sequencing works) o Understand principle of ddNTPs (no 3’ OH group so chain cannot be extended past them) 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 - - * 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 9 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: 10 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) 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 if this. This diagram is misleading because it seems like the processes are more separated than they are in reality. 11 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.) 12 Also called the anti-sense strand and the non-coding strand 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 I suggest picking one of these ways and sticking to it, personally I prefer 2 because it’s the sequence of the Non- easier and faster. template strand (coding strand or sense strand) and just swap the T’s with U’s 13 14 This diagram is a good illustration of how RNA is transcribed from DNA. In prokaryotes the growing RNA chain would be covered in ribosomes already translating it into protein even before it is detached from the DNA. In eukaryotes the 5’ cap is added as the chain is growing but the poly-A tail is added after the RNA dissociates from the DNA. The introns are also spliced out after the transcription process is done. 15 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. - 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. 16 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] I would know that 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). The figure that Dr. Baker put in his lecture slides is a bit too detailed for what you need to know in my opinion, as always check with him to figure out if you need to know something for sure or not. 17 Some introns are called ribozymes and they are able to splice themselves out of the RNA transcript without an external enzyme. I only put this figure in for demo purposes, just be aware that these self-splicing introns exist. Introns that are not self-splicing need the help of a “spliceosome” or snRNP complex. Which you should know the general process of, but no details necessary. 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). Know the basic process of how this could be accomplished in a lab setting. Microarray or DNA Chip Technology: You might have a multiple choice Q about this, but over-all just know it exists for now, no need to know any details. Regulatory RNAs: These are more important for you to understand a bit about how they work. They work by creating a double strand 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. 18 Know this general structure, but Amino Acids no need to memorize the specific structures of the 20 different amino acids. By convention, peptides are always written from N-terminus to C-terminus because they have directionality. The structure of a peptide bond is also very important to know. Ribosomes are the cellular machinery that translates mRNA into peptide chains. Ribosomes are composed of proteins and RNA in approximately 50:50. They are divided into a large subunit and a small subunit. Know the sizes of both subunits in prokaryotes and eukaryotes and know which is which.
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