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Table of Contents Things to study: .............................................................................................. Error! Bookmark not defined. Lecture Notes ..................................................................................................................................

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Table of Contents Things to study: .............................................................................................. Error! Bookmark not defined. Lecture Notes ................................................................................................................................................ 2 Lecture 21 11/13/18 Gene Regulation Part 2 ........................................................................................... 2 Lecture .................................................................................................................................................. 2 Big Picture ............................................................................................................................................. 5 Lecture 22 11/20/18 RNA World .............................................................................................................. 8 Lecture .................................................................................................................................................. 8 Big Picture ........................................................................................................................................... 10 Lecture 23 11/27/18 DNA Recombination & Transposons..................................................................... 13 Lecture ................................................................................................................................................ 13 Big Picture ........................................................................................................................................... 15 Lecture 24 11/29/18 Cell Junctions, Adhesion, and Extracellular Matrix............................................... 17 Lecture 25 12/4/18 Cell Cycle ................................................................................................................. 23 Lecture 26 12/6/18 Cell Cycle cont. ........................................................................................................ 26 Canvas Quizzes............................................................................................................................................ 30 Previous Exams ........................................................................................................................................... 31 Exam 1..................................................................................................................................................... 31 Multiple Choice ................................................................................................................................... 31 Short Answer....................................................................................................................................... 32 Exam 2..................................................................................................................................................... 32 Multiple choice ...................................................................................................................................32 Short answer ....................................................................................................................................... 33 Exam 3..................................................................................................................................................... 34 Multiple Choice ................................................................................................................................... 34 Short Answer....................................................................................................................................... 34

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Compare and contrast the bacterial CRISPR and eukaryotic RNAi Homologous recombination Three classes of transposable elements (similarities and differences) o Life cycle (DNA only or RNA intermediate) o Enzymes required Cell cycle checkpoints Cyclin Cell cycle checkpoints o Functions/locations

Lecture Notes Lecture 21 11/13/18 Gene Regulation Part 2 Lecture • Post-transcriptional Regulation o Transcriptional regulation is not the only level at which gene expression is regulated o Growing realization that most eukaryotic genes are regulated by alternative splicing ▪ A gene with even just a few exons can produce many different mRNAs via alternative splicing – “splice variants” ▪ Some variants may simply lack one or more exons ▪ Sometimes there are mutually exclusive alternative exons ▪ May be regulated by splicing repressors or splicing activators o Similar theme: alternative poly-A site addition o mRNA can also be regulated after processing is complete o HIV uses regulated nuclear export to allow RNA molecules containing some introns to be exported from the nucleus – important for the HIV life cycle o Cells can use regulated cytosolic localization to place specific mRNAs at specific locations in the cell o Allows mRNA, and the encoded protein, to be concentrated in a particular part of the cell o Some genes are regulated by mRNA stability – mRNA is rapidly degraded under certain conditions • Gene Silencing by Non-Coding RNAs o Short non-coding RNAs can regulate gene expression o MicroRNAs (miRNAs) are derived from precursors that fold into “hairpin” stem-loop structures o After processing, a short double-stranded RNA is generated and associates with a set of proteins to form an RNA-induced silencing complex (RISC) o One strand of the RNA is degraded, and the other makes base-pairing contacts with an mRNA target o Depending on the degree of base-pairing, the target mRNA may be degraded, or translation may be inhibited o Small inhibitory RNAs (siRNAs) mediate the process of RNA interference

Double-stranded RNA (dsRNA) is formed by base-pairing between complementary regions of separate RNA strands o dsRNA is cleaved by the Dicer nuclease to form short double-stranded RNAs: siRNA o As with miRNA, siRNA associates with proteins to form RISC, and target mRNAs are cleaved o siRNA can associate with a slightly different set of proteins to form an RNA-induced transcriptional silencing (RITS) complex, which inhibits gene transcription by modifying chromatin structure What advantages might come from post-transcriptional regulation? o Can respond to environmental stimuli more rapidly than transcriptional regulation Regulation of Protein Translation o Once an mRNA has been synthesized, protein amounts can still be regulated at the level of translation o Information in the 5’ and 3’ untranslated regions (UTRs) can regulate translation efficiency as well as mRNA stability ▪ 5’UTR RNA structure can allow binding of translation repressor protein that blocks ribosome access ▪ RNA structure itself (e.g. hairpin) may inhibit ribosome scanning ▪ “Riboswitch” structure may use binding of an ion or small molecule to switch between translation “on” and “off” states ▪ Repressors binding to 3’ UTR can prevent communication between 5’ and 3’ ends of mRNA (required for efficient translation initiation o Phosphorylation of initiation factor eIF2 can inhibit global protein synthesis ▪ eIF2 uses GTPase motif to mediate binding of initiator met-tRNA to small ribosomal subunit ▪ eIF2B is a GEF that catalyzes exchange of GDP for GTP, activating eIF2 ▪ Phosphorylation of eIF2 turns it into an inhibitor of eIF2B, blocking translation initiation o Context surrounding AUG can allow regulation by “upstream open reading frames” (uORFs) ▪ Open reading frame is a sequence starting with an AUG and ending with a stop codon, theoretically able to encode a polypeptide ▪ Some genes have short ORFs upstream of the “main” coding sequence – if the ribosome begins to translate a uORF, it will terminate with the stop codon and fall off the mRNA before reaching the main coding sequence ▪ Phosphorylation of eIF2 turns decreases global translation initiation, allowing some ribosomes to “read through” uORFs to reach the main coding sequence – a strategy to selectively increase a few proteins during stress conditions (e.g. amino acid starvation) uORFs Regulate Translation of ATF4 o ATF4 is a transcription factor involved in responses to various stresses, including amino acid starvation o Under non-stress condition, translation of ATF4 is inhibited by uORFs o Under stress condition, eIF2 is phosphorylated, reducing initiation at uORFs o

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o Some ribosomes can read through and initiate translation of ATF4 Regulation of Protein Translation o Internal ribosome entry site (IRES) allows ribosome to skip the first AUG by binding to an internal site. o This allows two different protein sequences to be derived from a single mRNA ▪ Different initiation sites may lead to skipping of a signal sequence (required for secreted/transmembrane proteins), and so switching between cytosolic and secreted forms of a protein ▪ IRES may sit between two separate ORFs, allowing independent simultaneous translation of two completely different proteins from one mRNA ▪ Viruses use IRES-initiated translation to synthesize viral proteins while inhibiting host cell cap-dependent translation Regulation of Protein Activity o As discussed previously, protein function is heavily regulated by post-translational modifications o Phosphorylation may directly affect protein activity, or may generate new binding sites for protein-protein interactions o Various post-translational modifications can direct proteins to new cellular sites Regulation of Protein Stability o Protein turnover is another point of regulation o Some proteins are highly stable, but some are rapidly degraded, and then resynthesized when needed o Damaged or misfolded proteins must be destroyed to prevent accumulation of malfunctioning proteins o The ubiquitin/proteasome system allows for regulated destruction of proteins ▪ Targeted protein is polyubiquitylated ▪ Proteasome recognizes polyubiquitylated protein and degrades it into short peptides o Stability of an undamaged protein can be controlled o “N-end rule”: Identity of N-terminal amino acid defines intrinsic stability ▪ N-terminal amino acid affects stability: Arg, Lys, His, Phe, Leu, Tyr, Trp, Ile, Asp, Glu, Asn, and Gln are destabilizing ▪ All proteins start with Met (stabilizing), but regulated cleavage of N-terminus can lead to degradation of protein ▪ One example: cleavage of inhibitory securin protein allows cell cycle progression into anaphase o Phosphorylation (or other post-translational) modifications can target a protein for ubiquitylation, and thus degradation o Proteasomal degradation is very energetically expensive ▪ Must synthesize multiple copies of ubiquitin ▪ Ubiquitin ligation uses 2 ATP equivalents (ATP -> AMP) for each added ubiquitin ▪ Unfoldases require ATP to feed protein into proteolytic cylinder o Other major cellular proteolytic organelle, lysosome, degrades proteins with no ATP requirement, but is non-selective

Highlights the importance of having a mechanism for regulated protein degradation – cell is “willing” to spend lots of energy Cells can use one DNA sequence to make multiple protein sequences. Which of the following does NOT illustrate this principle? o

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Big Picture • Although much of gene expression is regulated at the level of RNA transcription, additional levels provide rapid and dynamic control of protein level/activity o Alternative splicing ▪ Different combinations of exons can produce different mRNA splice variants • Optional exon; optional intron; mutually exclusive exons; internal splice site o Poly-A site addition ▪ By alternating which poly-A site will be used, you end up with different functions in the end ▪ Ex. gene that encodes for antibodies (made by B cells) • B cells can make two versions: one that is tethered to the plasma membrane of a cell (when tethered to surface of B cell, it is called a B cell receptor); If it lacks a transmembrane domain, the same protein can be secreted (secreted form is called an antibody) • Regulated by use of two different poly-A sites; first before M1 region and second after M2 region, in between is transmembrane domain sequence o If you splice at the first site, you don’t have the transmembrane domain region, so the protein gets secreted o If you bypass the first site and use the second site, now the transmembrane domain sequence is included and the protein will be retained on the surface of the cell as the B cell receptor o Regulated nuclear export allows RNA with some introns to be exported from the nucleus (ex. HIV) o Cells can target RNAs to specific subcellular localization or places within the cell to regulate their expression (ex. fruit fly development) ▪ Allows mRNA and the encoded protein to be concentrated in a particular part of the cell ▪ Initially, have one cell. Instead of one cell that divides, the nuclei divides. You have one cell with multiple nuclei, and RNAs are being expressed. Those RNAs get shuttled into distinct spots within the cytosol. Over the process of development, you end up creating plasma membrane divides in between these nuclei. Because the RNAs have been segregated into distinct subcellular localization spots within the cell, once the plasma membrane barriers form around the individual nuclei, now the cells have different identities and thus different functions o Some genes are regulated by mRNA stability – how long the RNA sticks around for may vary among different genes/transcripts ▪ Can stabilize mRNA using a protein

Ex. iron starvation: iron in bloodstream gets transported by transferrin. Iron is important but also can be toxin, so must be tightly regulated. Have a mRNA transcript made for transferrin. In order to get iron into cells, transferrin must bind to transferrin receptor (need more iron; upregulate transferrin receptor to grab more iron out of bloodstream). Aconitase protein likes to bind to iron. If cell has low levels of iron, aconitase will bind to a structure in the transferrin mRNA to stabilize the mRNA. There is no iron around so aconitase is not in association with iron but instead it binds and stabilizes the transcript that encodes the transferrin receptor. mRNA is stabilized and is around for a longer time, enabling more ribosomes to use the transcript to make more transferrin receptor protein. The receptors will go to the surface of the cell, grab more iron and bring it into the cell. There is now more iron in the cell, so aconitase will let go of the transferrin receptor mRNA and binds to iron. In releasing the transcript, the transcript becomes susceptible to nuclease cleavage and further degradation Small non-coding RNAs can regulate gene expression o Micro RNAs (miRNAs) ▪ Micro RNAs are derived from single RNA that fold into hairpin structures. This gets further processed and loaded into RISC protein complex and gets targeted to mRNA in cytoplasm that compliments its sequence. Depending on level of base pairing between the micro RNA and its target, it can result in degradation of mRNA or repression of translation ▪ In nucleus, the initial mRNA loops back on itself and is now base pairing with itself. Process of cropping cleaves off 3’ and 5’ ends of immature miRNA. This gets exported into cytoplasm, where Dicer enzyme cleaves off the loop at the end, leaving a short stretch of double stranded RNA. This gets loaded into RISC complex (composed of argonaute protein and other factors). One of the two RNA strands gets degraded/left behind; RISC complex now has the remaining strand, which can base pair with complimentary sequences on mRNA in the cytoplasm. • “Slicing”: RISC complex mediates cleavage of mRNA, rendering ends free and open for nuclease to further degrade the target mRNA because no cap or tail protecting ends • If there is some level of matching but not as extensive, then translation of the transcript is generally repressed, the RNA eventually get shuttled off to P-bodies and degraded as well through a different process o Small inhibitory RNAs (siRNAs) ▪ Mediates the process of RNA interference ▪ Process is very similar to miRNAs, big difference between processes is the origin of the double stranded RNAs • miRNA: single RNA strand that loops back on itself • siRNA: two separate RNA strands •

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Double stranded RNA (dsRNA) comes from two separate RNA strands that complement each other and can interact through base-pairing ▪ dsRNA is cleaved by Dicer nuclease to form short double-stranded RNAs (siRNAs). siRNAs associate with other proteins to form the RISC complex to target mRNAs for cleavage ▪ siRNAs can also associate with an RNA-induced transcriptional silencing (RITS) complex that modifies chromatin structure and impact gene transcription • Association with RITS complex can modify chromatin structure and impact transcriptional regulation (histone methylation, DNA methylation) ▪ siRNA pathway is thought to be an ancient anti-viral defense mechanism (viruses often produce dsRNA, but eukaryotic cell rarely do) As with RNA transcription, multiple mechanisms regulate protein translation at initiation step o Factors can interact with the untranslated regions of the transcript (3’ and 5’ UTRs) o Information in the 5’ and 3’ untranslated regions (UTRs) can regulate translation efficiency as well as mRNA stability ▪ 5’ UTR structure can allow binding of translation repressor protein that blocks ribosome access ▪ Repressors binding to 3’ UTR can prevent communication between 5’ and 3’ ends of mRNA, which is required for efficient translation initiation ▪ RNA structure itself can inhibit ribosome scanning (e.g. hairpin loop) ▪ “Riboswitch” structure can use binding of an ion or small molecule to switch between translation on/off states o Ex. iron starvation with ferritin mRNA o Regulate initiation factors that impact ribosome binding ▪ eIF2 mediates the binding of the initiating tRNA, required to start translation. eIF2B catalyzes the exchange of GDP for GTP, which activates eIF2. ▪ Phosphorylation of eIF2 turns it into an inhibitor of eIF2B, which blocks translation initiation ▪ Globally decreases protein translation initiation, but leads to the upregulation of translation of a small subset of RNAs o Use a sequence called an IRES (internal ribosome entry site) to skip the initial AUG ▪ Allows two different protein sequences to be derived from a single mRNA • binds to an internal site ▪ Not common in eukaryotes, but viruses use IRES-initiated translation Post-translational modifications regulate protein function: activation/de-activation, colocalization with interacting molecules, assembly into multi-protein complexes, etc. o Phosphorylation o Proteins can be directed to new cellular sites Protein level can be regulated via ubiquitin/proteasome-mediated degradation o Add ubiquitin onto a protein and feed that protein into the proteasome to be chewed up by proteases

Lecture 22 11/20/18 RNA World Lecture • Epigenetic regulation o “Epigenetic” inheritance is defined as any heritable difference that does not rely on changes in the DNA nucleotide sequence o This is the basis for cellular differentiation: liver cell division gives rise to more liver cells, without the cells having to “relearn” to be liver cells o Multiple mechanisms can contribute to epigenetic changes ▪ Stable expression of a regulatory protein via a positive-feedback loop • Once protein A is made, it maintains its own expression. This kind of positive feedback loop thus provides a stable phenotype • Same transcription factor regulates its own expression, once TF is turned on it stays on (turned off by specific signals, etc.) ▪ Covalent modification to histones, changing chromatin state • Covalent modification of histone recruits enzymes that replicate the same “histone code” when a cell divides, maintaining chromatin structure in daughter cells ▪ Methylation of NDA on cytosine residues • Methylation of cytosine in CG sequences suppresses gene transcription. Maintenance methyltransferase, methylates CG sequences that are already paired with methylated CG, allowing the methylation pattern to be maintained during DNA replication...