Cellular Biology synthesis, Summaries of Cellular and Molecular Biology

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M. Jones, G. Jones,

Cellular Biology

Table of Contents

• Lecture Notes Things to study Error! Bookmark not defined.
  • Lecture 21 11/13/18 Gene Regulation Part
    • Lecture
    • Big Picture
  • Lecture 22 11/20/18 RNA World
    • Lecture
    • Big Picture
  • Lecture 23 11/27/18 DNA Recombination & Transposons
    • Lecture
    • Big Picture
  • Lecture 24 11/29/18 Cell Junctions, Adhesion, and Extracellular Matrix
  • Lecture 25 12/4/18 Cell Cycle
  • Lecture 26 12/6/18 Cell Cycle cont.
• Canvas Quizzes
• Previous Exams
  • Exam
    • Multiple Choice
    • Short Answer
  • Exam
    • Multiple choice.....................................................................................................................................
    • Short answer
  • Exam
    • Multiple Choice
    • Short Answer

o 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 eIF ▪ 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 ATF 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 Some ribosomes can read through and initiate translation of ATF

• 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

• 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

▪ 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, co- localization 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

o Can bring distant parts of RNA together o Can form quartnerary structures o RNAs can never be as complex as proteins because there are only 4 nucleotides to work with while proteins have 26 amino acids

• Ribozymes are RNA enzymes that can catalyze chemical reactions – e.g. specific RNA cleavage. o Ribozymes: RNAs folded up such that they have catalytic function
• Ribozymes can be subjected to allosteric regulation by the cooperative binding of two small molecules o Function can be directed by the binding of small molecules outside of the catalytic site o Ex. from slide 13: Binding of FMN AND theophylline are required for self cutting
• We can make/select Ribozymes that catalyze a reaction o How to generate an RNA that has kinase activity? o Large pool of double-stranded DNA molecules, each with a different, randomly generated nucleotide sequence o Transcription by RNA pol and folding of RNA molecules o → Large pool of single-stranded RNA molecules, each with a different, randomly generated nucleotide sequence o Addition of ATP derivative containing a sulfur in place of an oxygen o → Only the rare RNA molecules able to phosphorylate themselves incorporate sulfur
• RNA can perform a variety of chemical reactions
• Catalysis & Replication: RNA that could catalyze its own synthesis o RNA replication was likely a key requirement for progressively making more complex collections of molecules
• RNA polymerase made of RNA can have > 95% accuracy o Ribozyme RNA polymerases that we have made (like one above) can only make ~ nucleotide long RNA
• TNA (threose nucleic acid; 4 - carbon) and PNA (peptide nucleic acid) would have been more stable molecules that could perform the roles proposed for RNA
• Origin of life according to the RNA World Hypothesis – ‘Replication first’
• Lipid World hypothesis – ‘Compartmentalization first’ o The chemistry of life began around a bilayer o Need to keep molecules from diffusing away
• Life first harnessed energy – ‘Metabolism first’ o A metabolic cycle arose near a geothermal vent. o Life uses a proton gradient to make ATP. o Life formed somewhere with a natural proton gradient.
• Why not have it all? – ‘Everything first’ o Life originated in shallow lakes near geothermally active areas or meteorite impact zones. o The protocell had a membrane enclosure that housed RNA-like enzymes, and harnessed energy using proton gradients.
• Which of the following supports the RNA world hypothesis for the origin of life? The existence of natural ribozymes

• Experiments in the worm C. elegans were key to uncovering many forms of RNA-directed gene regulation
• Ease of delivery and potency of silencing have enabled genome-wide RNAi screens to determine gene function o Double-stranded RNA can be expressed in bacteria that are then fed to worms. o Double-stranded RNA can be made in vitro and then injected anywhere in the worm.
• RNA interference : Double-stranded RNA can potently trigger the silencing of genes of matching sequence
• micro RNAs: Genes that encode short RNA that control the stability and translation of mRNA
• So how does something that is only 20 - 22nt long affect other genes? They target the mRNA transcripts and utilize cellular machinery that normally degrades RNAs. o mRNA can be degraded from both ends once the Cap and poly-A are removed
• RNA interference is used for gene regulation in many plants and animals o Cleavage of target RNA o Translational repression and eventual destruction of target RNA o Formation of heterochromatin on DNA from which target RNA is being transcribed
• Argonaute/Piwi proteins can act as “slicers” of RNA o Slicing: generate short stretches of RNA, degrade complimentary strand to leave one strand, some portion of RNA molecule with nucleotides available to interact with some target RNA and that RNA will be cut by the argonaute/piwi protein
• micro RNAs control the stability and translation of messenger RNA (mRNA) o RISC = R NA I nduced S ilencing C omplex
• Double-stranded RNA triggers multiple gene silencing mechanisms o R NA I nduced S ilencing C omplex o R NA I nduced T ranscriptional S ilencing Big Picture
• The RNA world is a hypothesis for the origin of life on earth. But, it still is an RNA’s world – RNA regulates many processes in current organisms o The structural, functional, and information storage capabilities of RNA suggest that early life may have consisted of many RNA machines ▪ RNA can form secondary and tertiary structures
• Secondary: single strand, double strand, single-nucleotide bulge, three- nucleotide bulge, hairpin loop, symmetric internal loop, asymmetric internal loop, two-stem junction or coaxial stack, three-stem junction, four-stem junction o Imperfect pairing, nonpaired nucleotides, RNA molecule folds on itself, symmetrical non base-pairing, multiple strands interact with each other
• Secondary structures can interact with each other to form tertiary structures o Unpaired nucleotides can pair with other unpaired nucleotides on other RNA molecules
• Tertiary: pseudoknot, kissing hairpins, hairpin loop-bulge contact

▪ Life may have evolved where there was a natural chemical gradient and became organized around that o Why not have it all? – Everything first ▪ Things were happening simultaneously, what drove life was the coincidental concordance of all of these things happening in a single place

• There was a natural proton gradient/electrochemical gradient, there were lipids that were able to compartmentalize, the compartmentalization captured some of the catalytic RNA that was arising independently – all of these events occur simultaneously in the same location (not sequentially)
• Small RNAs that can regulate genes are found in most organisms including bacteria, plants, and animals. o RNA interference: double-stranded RNA can potently trigger the silencing of genes of matching sequence ▪ Used for gene regulation in many plants and animals
• Have a stretch of double-stranded RNA (from two separate transcripts with complimentary regions) that interacts with a series of proteins (argonaute/piwi proteins - act as “slicers” of RNA; both bind to double- stranded RNA and allow it to be processed to leave a short stretch of 20 - 22 nucleotides long) one strand gets degraded and the other is used as a targeting strand to find other complimentary sequences in other RNA molecules
• In some cases, when they interact, RNA molecule is cleaved. Other cases, you get translational repression. If a RNA molecule sits like this for long, it can be targeted for destruction by other mechanisms. The RNA molecule can also interact with DNA sequences and lead to changes in chromatin structure o microRNAs: genes that encode short RNA that control the stability and translation of mRNA ▪ control the stability and translation of mRNA o The small RNAs target the mRNA transcripts and use cellular machinery that normally degrades RNAs ▪ mRNA can be degraded from both ends once the cap and tail are removed
• Can cleave off 5’ cap, cleave off 3’ tail, or cleave in middle to produce two molecules missing either the cap or the tail
• RNA-directed immune surveillance occurs in bacteria, invertebrates and possibly vertebrates. o Bacteria use small non-coding RNAs to protect themselves from viruses ▪ Short viral DNA sequence is integrated into CRISPR locus ▪ RNA is transcribed from CRISPR locus, processed, and bound to Cas protein (DNA-cleaving enzyme) ▪ Small crRNA in complex with Cas seeks out and destroys viral sequences
• RNA-directed histone modifications, DNA methylation, and even DNA rearrangements have been observed, suggesting that RNAs can ultimately trigger these epigenetic inheritance mechanisms.

• RNAs can guide the cutting of any DNA sequence in almost any organism in CRISPR-based genome editing o The Cas9 enzyme can be “programmed” by a guide RNA to cut a specific DNA sequence ▪ There is a one-to-one correspondence with RNA-RNA. That means if you have a specific sequence of your guide RNA for the CRIPSR RNA, then you can simply change the sequence and now you change the target RNA. You can generate a CRISPR RNA/guide RNA that would attack any sequence of nucleic acids that you want ▪ All we have to do is generate a guide RNA that has a specific sequence that will target a specific double stranded DNA and cleave it o The Cas9 enzyme can be modified for sequence-specific genome editing and gene regulation ▪ Instead of introducing a double stranded break, we can modify the Cas enzyme so that it will bind to DNA but won’t cause a double stranded break. If the Cas enzyme is engineered so that in addition to having an inactive cleavage site, we have the activation domain from some regular transcription factor. Now we have a precise sequence that we know we can get this to bind to, and we have an activation/suppressor domain (anything to regulate gene expression). We can now very easily generate a sequence-specific transcription factor for any sequence

Lecture 23 11/27/18 DNA Recombination & Transposons

Lecture

• Bacteria use small non-coding RNAs to protect themselves from viruses o CRISPR: C lustered R egularly I nterspaced S hort P alindromic R epeats o Step 1: short viral DNA sequence is integrated into CRISPR locus o Step 2: RNA is transcribed from CRISPR locus, processed, and bound to Cas protein o Step 3: small crRNA in complex with Cas seeks out and destroys viral sequences
• Bacterial CRISPR and eukaryotic RNAi share some similarities. Which of the following is NOT an analogous pair? Long dsRNA and target mRNA.
• Compare and contrast the Cas/CRISPR system with the RNAi system
• CRISPR : The Cas9 enzyme can be “programmed” by a guide RNA to cut a specific DNA sequence o PAM = P rotospacer A djacent M otif (typically NGG) o Guide RNA attacks any sequence of nucleic acids as desired ▪ Don’t need to rely on CRISPR locus for targets o Generate a guide RNA with a specific sequence that will target a specific DNA sequence and cleave it
• Double-stranded breaks made by Cas9 are repaired to introduce random deletions or precise mutations o Nonhomologous end joining ▪ Loss of nucleotides due to degradation from ends ▪ End joining ▪ Deletion of DNA sequence o Homologous recombination

o The branch point is equally likely to migrate from left to right as it is to migrate from right to left. o Specialized helicases and the expenditure of energy can provide directionality to branch migration. o In some situations, DNA polymerase can also drive the directional branch migration.

• Homologous recombination is also used to flawlessly repair double-strand breaks o The first step in homologous recombination is 5’ to 3’ degradation by an exonuclease at the double strand break region. o This generates free 3’ ends that can invade homologous DNA and through branch point migration result in the synthesis of new DNA. o DNA polymerase drives this branch migration. o Strand invasion by any one free 3’ end is sufficient for homologous recombination to occur.
• Homologous recombination in meiosis can generate crossovers o During meiosis, specific nucleases can trigger a double-strand break to facilitate DNA mixing during sexual reproduction o Mixing occurs through the creation of a double holliday junction and is resolved into a conversion or a crossover. o Crossovers are rarer than non-crossovers Big Picture
• Cells and organisms use homologous DNA recombination to fix, rearrange, or mutate their genomic DNA through strand invasion, branch migration, and DNA synthesis and repair. o Homologous recombination is used to fix broken replication forks ▪ If you have a single stranded DNA nick, what happens is as the replication fork progresses and the nick isn’t sealed, then when the replication fork hits the nick, the piece of DNA will fall off and we lose the fork. Nothing past the nick gets replicated ▪ First step: we have an exonuclease that degrades the DNA (5’ to 3’) that was part of the fork that was broken. This leaves a 3’ overhang that invades the existing double helix; it base pairs with the homologous strand and displaces the existing homologous strand. The invasion is repaired by breaking the homologous strand and synthesis continues from the invading strand. ▪ Degradation of one strand to leave a 3’ overhang, the 3’ overhang invades the duplex of the homologous strand. The replication fork moves to where the homologous pairing occurs, then the second strand is broken and DNA is repaired through general repair processes. We can then progress through the rest of the replication fork ▪ 3’ end invasion depends on the ability of DNA sequences to base pair by random collision
• RecA/Rad51 protein facilitates strand invasion o Binds to double stranded portion and allows a triple strand to form o Homologous recombination is also used to flawlessly repair double-strand breaks ▪ With the double strand break, the cell doesn’t know if any nucleotides were lost. If any were lost, then a sequence of DNA would be lost

▪ We get degradation of the ends, 3’ overhang, and we have two chromosomes at every position. If a double strand break occurs on one chromosome, we use the other chromosome as a template to fix the broken one. We get strand invasion, then synthesis of the new strand of DNA. We can now separate the strand and put it back to its original chromosome. We can remove the invading strand, match it up to the original chromosome and its pair, bridging the break, DNA polymerase can restore the sequence in between and ligase can seal it up. The template strand is left intact, as the invasion was temporary o Homologous recombination in meiosis can generate crossovers ▪ These are programmed DNA breaks. Enzymes bind to one chromosome and cuts it, leaving a double strand break. We get the overhangs, but instead of one strand invading the sister DNA, we have double invasion (both strands are going to break). this leads to a structure called the Holliday junction. There is a second cleavage event that is resolved either by the strands going back to where they were originally, or the DNA pieces can swap positions, exchanging DNA between chromosomes o Conservative site-specific recombination can reversibly rearrange DNA ▪ Instead of a crossover event, with a circular DNA we can integrate it into the DNA through homologous recombination

• Transposons use similar mechanisms to become parasites on genomic DNA and to move from one place in the genome to another. In this way transposon and transposon remnants have now become ~50% of our genome. o There are three major classes of transposable elements (retro: RNA-based genomes that get converted into DNA and then integrated into the genome) ▪ DNA-only transposons - Short inverted repeats at each end - Moves as DNA, either by cut-and-paste or replicative pathways - Transposase required for movement ▪ Retroviral-like retrotransposons - Directly repeated long terminal repeats (LTRs) at each end - Requires reverse transcriptase and integrase o You have an intact double stranded piece of DNA that gets inserted in the DNA - Moves via an RNA intermediate produced by a promoter in the LTR ▪ Nonretroviral retrotransposons - Poly A tail at 3’ end of RNA transcript, 5’ end is often truncated - Reverse transcriptase and endonuclease o The reverse transcription happens using the nicked DNA as a primer and occurs at that site, no free double stranded DNA piece that gets integrated in - Moves via an RNA intermediate that is often produced from a neighboring promoter o DNA-only transposons in bacteria can move antibiotic resistance genes from one place to another ▪ Sequence homology between short inverted repeats are used for excision ▪ DNA moves as a protein-DNA complex (transposase dimer + excised DNA)

• DNA-only transposons in bacteria can move antibiotic resistance genes from one place to another o DNA-only transposons contribute to the spread of antibiotic resistance. o At a minimum, they comprise of a transposase and flanking transposase recognition sequences
• DNA-only transposons can move using a cut-and-paste mechanism o Sequence homology between short inverted repeats are used for excision o DNA moves as a protein-DNA complex (transposase dimer + excised DNA) o Insertion results in flanking direct repeats
• Retroviral-like retrotransposons move using mechanisms that share similarities with retroviral life cycle (e.g. HIV) o Reverse transcription & Integration are key steps in how a retrovirus gains entry into the host genome. o Unlike retroviruses, retroviral-like retrotransposons do not encode capsid and envelope proteins
• Retroviral-like retrotransposons rely on a reverse transcriptase to convert transcribed RNA into DNA o RNA-dependent DNA polymerase, uses RNA template, no requirement for primer, has o Reverse transcriptase enzyme looks like a DNA polymerase with an attached RNAse H domain. ▪ Degrades RNA portion of RNA-DNA hybrid ▪ Enzyme can also act as DNA-dependent DNA polymerase; can replicate DNA strand running in opposite direction o RNA is degraded in the 3’ to 5’ direction and DNA is synthesized in the 5’ to 3’ direction.
• Retroviral-like retrotransposons rely on an integrase to insert their DNA into a new location o An integrase inserts DNA into the genome but unlike a transposase cannot catalyze excision. Thus, retrotransposons must move through an RNA intermediate.
• Nonretroviral retrotransposons rely on an endonuclease reverse transcriptase to move o This type of retrotransposon makes up a large fraction of the human genome. o Some elements (e.g. L1 and Alu ) are present in >1 million copies! Copies in new genomic locations arise in every 100 - 200 human births. o Relies on nearby promoters for RNA synthesis. o Relies on an cleavage of one of the DNA strands by an endonuclease that can bind the RNA and is attached to reverse transcriptase. o Reverse transcription occurs through the use of a DNA primer in the 5’ to 3’ direction from the nick generated by the endonuclease. o This new copy will again rely on nearby promoters to make RNA and the cycle continues.
• Which of the following mobile elements from Drosophila is not a DNA-only transposon? o Copia elements; these have directly repeated long terminal repeats and move with the help of a reverse transcriptase and an integrase
• Repression of transposable elements requires the PIWI subfamily of Argonaute proteins o P - element i nduced wi mpy testes (PIWI) proteins were first identified in Drosophila o In all these cases, PIWI proteins interact with a class of small RNAs called piRNAs ( p iwi- i nteracting RNAs)
• P-elements (a class of transposons) are repressed by RNAs deposited in the egg by the mother

o piRNAs are inherited through the egg. If a transposon ( P ) is present in the maternal side, the piRNAs made can repress the transposon. If a new transposon is only in the paternal side, it will remain active because the piRNAs inherited from the maternal side will not silence the new transposon.

• P-elements are repressed by RNAs deposited in the egg by the mother o As a result of this asymmetry in the inheritance of piRNAs, animals of the same genotype can differ based on the direction of the cross that generated them.
• Cells are organized into tissues in animals and plants
• Connective tissues and epithelial tissues are two ways in which animal cells are bound together o In epithelial tissues, the cells are directly next to each other, can resist stress because can be attached to ECM (basal lamina) o In connective tissues, cells are embedded in a matrix, the matrix bears most of the extracellular stresses o Fibroblasts in the connective tissue make and secrete the extracellular matrix material
• Four classes of cell-cell junctions can be distinguished based on distinct function & proteins involved o Anchoring junctions: Cell-cell or cell-matrix adhesion ▪ Cadherin or Integrin proteins are required for the two functions. o Occluding junctions: Permeability barrier ▪ Barrier functions ▪ Claudin proteins are required for function. o Channel-forming junctions: Intercellular passages ▪ Connexin or innexin proteins are required for function. ▪ Allow small ions to move from one cell to another ▪ Gap junctions o Signal-relaying junctions: Transmit signals ▪ Anchorage and signaling proteins are required for function. ▪ Allow secreted material from one cell to be recognized by another ▪ Complicated junctions, have proteins that are involved in holding the cell together as well as signal transduction machinery
• Transmembrane proteins link the cytoskeleton to extracellular structures o Cell-to-cell: Cadherins and integrins o Cell-to-matrix: Integrins o Connections between the cytoskeletal filaments and integrins/cadherins occur indirectly via intracellular anchor proteins. ▪ Can be attached/detached through anchor protein regulation
• Cadherins use Ca2+^ and homophilic binding to connect cells together o Cadherins: calcium dependent adherence proteins, not adherent in absence of Calcium and vice versa o Homophilic → interactions between the same proteins. o Extracellular calcium is used to trigger conformational changes in the cadherin molecule. o Thus, Cadherin molecules in two cells can interact with each other in a regulated manner.
• Homophilic adhesive properties of different cadherins can sort cells into “tissues” during development