BIOL 3010: Study Questions

Genetics

What are some of the major technological advances that have facilitated genetic and genomic analyses since ~1970?

- DNA sequencing 1977 with the Sanger method
- invention of PCR by Kary Mullis in 1983
- Humane genome sequenced 1986-2000
- targeted genome editing 2010 (CRISPR)

Explain how PCR works.

-DNA strands are seperated by denaturing at high temperatures, this breaks the H bonds holding the strands together
-the DNA is cooled and primers are added to each of the strands at the 3' ends, called annealing
-the temperature is increased slightly and extension occurs as the primers move along the strands
-this process repeated Manny times to get Manny copies of the same DNA

What is a gene? What is an allele?

gene: segments of DNA found on chromosomes
allele: different forms of a gene

Darwin described the process of natural selection. What is needed for this process to occur?

1. variation - individuals among a population differ in some trait
2. fitness differences: consistent relationship between value of a trait and reproductive success
3. inheritance - consistent relationship for value of a trait between parent and offspring

What are Mendel's "Laws" of Segregation and Independent Assortment and what analyses led him to these laws?

Law of segregation: two alleles from each parent segregate independently of one another during gamete formation
Law of independent assortment: different pairs of alleles (for diff genes) segregate independently of one another during gamete formation
observed this using the sweet pea, saw that color and shape assorted independently of one another

additivity

A mechanism of quantitative inheritance such that the combined effects of genetic alleles at two or more gene loci are equal to the sum of their individual effects.

continuous vs discrete traits

continuous variation shows an unbroken range of phenotypes of a particular character in the population whereas discontinuous variation shows two or more separate forms of a character in the population, A discrete trait is a phenotype that manifests as clear and separable differences in a population, while a continuous trait is a phenotype that manifests as a continuum along a spectrum. Examples of discrete traits are dimples and albinism. Examples of continuous traits are height and eye color.

Describe the basic structure of nucleotides and DNA or RNA.

DNA:
Nucleotides: A, G, C, T
deoxyribose sugar
phosphate group
RNA:
Nucleotides: A, G, C, U
ribose sugar
phosphate group
phosphodiester bonds link nucleotides
- DNA structure: double helix with

What observations suggested that particular bases pair in DNA?

A:T and G:C were seen in the same ratio in the base pairing of DNA across Many organisms, Chargaff

What are examples of chemicals that interact with specific features of the DNA helix?

-DAPI stain -- affinity for DNA and intercalates into minor groove, fluoresces and allows detection of where DNA is
- furanocoumarins (psoralen) --- chemical from giant hogweed, contacts skin and when exposed to UV will cause cross linking, this causes issues in DNA replication and leads to cell death, causes wounds in epidermis

Define "life," at least according to NASA.

"a self sustaining chemical system capable of darwinian selection"

What are the essential features of a genetic material and what makes RNA a particularly good candidate for being the first one?

Genetic material must be able to: store information, express information, replicate, and accommodate introduction of new variation
RNA is able to:
- encode information
- complex folding patterns --- single strand can fold on itself to make double stranded areas, shows may have some function due to structure
- highly conserved across all forms of life --- some individual types found across all forms of life, some rRNAs in ALL kingdoms of life
- can act as an enzyme, ribosyme --- ribosomal RNA, ribonuclease-P cleaves phosphodietster bonds on tRNA and other small RNAS, can be isolated and work without proteins!, self splicing introns are noncoding regions spliced out without any proteins
- self replicating --- not currently in living organisms but in viruses

What observations and experimental evidence suggest that self-replicating ribozymes might have existed in nature?

- extant ribozymes do not have replicase activity needed for RNA world but RNA polymerase ribozymes have been selected for in lab to add 1 to 3 nucleotides at a time
- able to synthesize a 6 nucleotide long segment but not able to do one with hairpin folding structures
-after six rounds of selection ribozyme able to get through 3 nucleotides at a time and 2nd structures
- RNA world:
1. RNA molecules w/ catalytic activités assembled from nucleotide soup
2. RNA molecules evolve and diversify by self rep and mutations and recombination to provide materials for selection
3. RNA molescules begin to synthesize proteins first by making adapter RNAs to bind to activated amino acids, which then improve on ribozyme only functions
4. DNA appears w double strand as more stable way to hold genetic info and error correction

Are there any obstacles to the RNA World hypothesis, and, if so, how might they be accommodated?

Problems:
-each nucleotide has three chemical moeities
- contemporary nucleotides won't couple w/o chemical activation
- phosphates very limited
- adenine may be prevelant but not others
-different cations
-non biotic synthesis of ribose unlikely and ribose unstable
- solvent properties: nucleotides dont self assemble w h bonds in h20, polymerase RNA unstable in H20
-natural RNa polymerase ribozymes unknown
Accommodations:
- could have had reasonable substitutes to bases and materials needed (other sugars, linkers, solvents, and cofactors)
- likely RNA didn't look as it does today

What recent data suggest that an RNA-Peptide World might have occurred sooner than originally imagined?

RNA synthesis of peptides
- tRNAs have modified bases, critical AA w modified side chains added, one of modified bases interacts w AA and can couple and cleaned and dissociate (goes from 1AA peptide to 2 AA peptide)
- able to occur in vitro w only RNA chains
- contemporary tRNAS may have modified nucleoside bases thought to be relics of ancient RNA world
- modified bases can allow transfer and coupling of AA into peptide chain

What early evidence showed that genetic material could be transferred between organisms, and that such material is DNA?

-experiments with Streptococcus bacteria
- two forms: s form with polysaccharide coat and R form is individual
- s form = death and r form = alive
- spontaneous change between the two forms s can become r
- if s heat killed and then introduced w live r then the mice died, r was converted to the s form
- called TRANSFORMATION: genetic info from dead cells transferred to live cells

Explain the experimental results that led to the "one gene, one enzyme" hypothesis. What modern genetic and genomic data suggest the original hypothesis was framed too narrowly?

Beelte and Tatum in the 1940s
- screen for yeast that are unable to synthesize their own amino acids, used x rays to break DNA apart and let it rebuild itself, when rebuilt had mutations, could look for mutants that were unable to synthesize their own amino acids
- wild type can synthesize all of own amino acids
- took spores and used x rays then crossed to another wild type and got haploid spores
-grew on full medium and then grew on minimal medium
- the ones that died on minimal where then grown on individual amino acid mediums to see if survived
- certain example only survived when given arg so arg deficient
- found four different mutants with different issues in the steps to synthesize arg (missing certain enzymes, etc.)
-implies that genes code for proteins w distinct enzymatic actives and function in linear pathways
-NEW DATA: SHOWS GENES ARE COLINEAR
- high res mapping shows how induction of mutations altered amino acids in corresponding portions of proteins, missense mutants if changes AA, nonsense mutants if stopped transcription, each nucleotide associated w identity of one aa, multiple nucleotides for one aa because adjacent mutations could affect, mapped how genes go to proteins

What are the fundamental properties of the genetic code and how were they "deciphered"?

- was deciphered by making sythetic RNAs with only certain base composition, started with only U and only synthesized Phe, tried with diucleotides ad got two alternating aa, tried with three ad got the same, tried with four ad got four different Ones or a stop codon
- each AA is encoded by a codon of three nucleotides
- degeneracy -- diff codos can code for same aa
- three stop "non sense" codons
-initiatio code (AUG) marks the start of the reading frame
- mutations can alter the message ( missennse - change aa, nonsense - stop, silent - doest change aa, frameshift - changes entire sequence of protein
- DNA: two strands template is antisense and noncoding, RNA-like strand is sense and coding
- polarities same, 5' end of RNA makes the N terminal end of proteins

Define the different elements comprising a eukaryotic "gene body" as defined in class. How is this different from prokaryotes?

-cis regulatory elements (enhancers): locations on the gene for transcription factors to bind, can be upstream downstream or In the center of gene body
- core promotor: where RNA poly attaches ad forms complex
-TSS: where tells to start making RNA
- 5' UTR: where tells to start making RNA
- Start codon: where transcription begins and ribosome actually makes RNA
- exon: coding sequence actually in the final mRNA
-introns: noncoding sequence spliced out in mature mRNA
-stop codon: tells to stop synthesizing RNA
-3' UTR and transcription termination signal: tells machinery to stop making RNA
-"gene body" composed of irons ad exons and sometimes cis regulatory
- prokaryotes have no cis regulatory or 5'UTR or 3'UTR, also have no introns, have leader sequence instead of 5'UTR

Does a gene consist of more than its gene body?

yes, eukaryotic genes have a core promotor
also cis regulatory elements are upstream and downstream of the gene body

Compare exons and introns. Which have coding sequence and which have untranslated regions? Which appear in mature mRNAs?

-exons: coding sequence of DNA, have 5' and 3' UTR, appear in mature mRNA
-introns: noncoding sequence of DNA, spliced out in transcriptional processing

What is a cis-regulatory element? Can these elements always be identified computationally? Where do they reside in the DNA?

cis regulatory elements: short strands of DNA that serve as binding sites for transcription factor proteins, around 4 to 15 nucleotides long
- can be upstream, within introns, or downstream of gene, gene can fold so will be close at time of transcription
- dont often know where they are or what their significance is

start codon

AUG or ATG

stop codons

TAA, TAG, TGA

5' UTR

critical for recruitment of ribosome to mRNA for translation provide stability, post transcriptional modification of the gene including transport, stability, ad localization

TSS

location where the first DNA nucleotide is transcribed into RNA

core promoter

where transcription initiated ad RNA poly assembles

enhancers

cis regulator bound by activators, help to increase transcription of target genes

How does "Sanger sequencing" work and what modifications to the original method contributed to increased efficiency (and safety)?

- p32 radioactive nucleotide was put on the end of each of the primers for extension and then visualization after electrophoresis
- ddNTPS were added for each of the nucleotides one at a time, created different sized fragments bc cleaved at the base
- fragments were separated by size on a gel and exposed to film to see radioactive p32
- this was done for all four nucleotides with the four ddNTPS
-multiple reactions with radioactive nucleotides and each read about 100-200 nucleotides long
- advances: fluorescent ddNTPS, could add all the the reaction at the same time and avoided having to use radiation, run through capillary with matrix to separate by size and use a detector
- gave electropharogram which sep by diff in color

Sanger sequencing is sometimes referred to as "first generation" sequencing, whereas several methods of "second generation" (sometimes referred to as "next generation") sequencing have been devised. Currently the most commonly used of these is Illumina Corporation's Solexa sequencing. In what ways does this method differ from Sanger sequencing?

- uses a flow cell, genome is fragmented and primers are added to the ends of the DNA strands which cause them to stick to the cell, unlabeled nucleotides and polymerase added so DNA will form a bridge and make the complimentary strand, then are denatures to separate the two strands, nucleotides labeled with fluorescence and fluoropore stops and polymerase and primers are added, each complimentary strand is sequences one nucleotide at a time ad the stops, can use fluoresce to see the nucleus added, termination reversible and second added, can do this for 300-400 long and can do millions at a time on one chip

What are the steps typically used when attempting to generate a high quality "reference" genome sequence? What is the meaning of sequencing "coverage" and why is it important to sequence more DNA than is found in an individual genome?

- clone by clone sequecing is best method: genome is fragmented into large pieces and then put In bacterial artificial chromosomes as an intermediate, the clones are then assembled in order using the ends of the sequence or DNA fingerprinting, then the clones are each broken into small pieces one at a time and sequenced, fragments of clones are ordered, sequences of overlapping clones established to get reference sequence
-sequence coverage is important because must be sequenced many times to ensure all covered, some fragments rarer than others, "rain drops on sidewalk",. no every nucleotide hit on first sequence, have to do entire genome 50x to get good coverage

Why is sequence read assembly complicated by sequences that are identical or nearly identical at different places in the genome?

-hard to tell if these are duplicate copies or if there actually are two of the same sequence in different places, also may have alignments with slightly different parts that both could work and you dont know which one is correct

"Third generation" sequencing was not discussed in class but allows much longer sequencing reads than Illumina sequencing (e.g., 10 kb vs 150 bp per read), though at lower "throughput" and sometimes with less precision than Illumina. ("PacBio" is one example of third generation sequencing.) These newer technologies have eliminated the need to make bacterial artificial chromosome (BAC) libraries, as was done for the first draft of the human genome, and "new" genomes are often now sequenced and assembled computationally with a combination of second and third generation strategies. Why is it valuable to have an intermediate level of structure to one's sequencing effort, whether provided by long-read third generation sequencing, or second generation sequencing using BACs as the starting material? Why might third generation approaches now be preferred to BACs?

-intermediate levels of structure help to constrain to a relative area of where in the genome you are, you at least know how close together the sequence fragments are
- third gen may be more favorable because it eliminates another step which could lead to error by making clones In BAC, it also makes it quicker because you dont have to first order BACs before splitting them apart individually to sequence each one

dNTP vs ddNTP

dNTP - building blocks of DNA, have an OH on the 3' end
ddNTP - used to stop DNA polymerase, chain terminating, missing the OH on 3', used in Sanger sequencing

electropherogram

used in sanger method with fluorescent dyes, separates based one color of labeled nucleotides

capillary sequencer

used with sanger w fluorescent dyes, separated by sized and has detector to show flourescence

dye terminator

used in illumina flow cell method, after each dye labeled nucleotide is added the DNA synthesis is stopped and can be detected which was added, then is reversed so can continue

Early estimates of the gene number varied widely. What do whole genome sequences that have been annotated for genes tell us about actual numbers of genes, and what kinds of genes they are?

- total size: 3.6 Gb
- protein coding: 20,376
- 40,000 total gees
- gene body length: average 26,288 bp
- noncoding genes: 22,000
-exons per transcript: 7
introns per transcript: 6
mRNA- 2787 bp
-pseudogenes - ab 13,000, used to be functional but aren't currently
- shows much variation in size and composition, not only made of protein coding genes

Protein coding sequence comprises only about 1% of the genome. What is the rest?

introns, 5'UTR and 3'UTR, promoters, regulatory elemets, simple sequence repeats (ab 50% genome), transposable elements (encode proteins when functional which allow them to move in genome ad to copy themselves in genome, some may obtain stop codons and lose function, humans have ab 44%)

There are several types of repeated sequence in human and other genomes. What are their broad categories (recognizing that we will discuss in more detail later in the semester)?

1. Simple sequence repeats (SSR) - example are CA microsatelites, one SSR per 2kb, Manny people have different sizes so can use to determine paternity, different sizes because repetitive sequence so DNA polymerase can jump ahead or miss or add a sequence, might find several sizes of repeat in PCr
2. transposable elements: can encode proteins when functional which allow them to move within the gene and copy themselves within the genome, varies across species humans 44%

Why are microsatellites often variable in length? Are there usually consequences of such length variation for the organism?Why or why not?

micro satellites vary in length because they are repetitive so errors in DNA replication may occur, DNA poly may miss one or add one to the sequence, usually noncoding sequences so mutations typically have no affect on the organism, may people can have different length micro satellites, may different alleles (variants of a locus) for SSR repeat micro satellites

Define "locus" and "allele." What do these terms mean when applied to non-protein coding DNA (like microsatellites)?

Locus: the site or location of a gene on a chromosome
allele: one or more alternatives of a form of a gee
- can use alleles to describe the different lengths of SSR of micro satellites, can use alleles to describe the variants of a locus of micro satellites, can vary from person to person

Genome browsers like Ensembl , NCBI or UCSC provide a wealth of information. What are some of the features that can be readily accessed using such a browser? Do these browsers indicate every functionally important part of a gene?

can show 5'UTR and 3'UTR, can show exons and introns, can show the AI number used to look up the sequence, show the q number identifing position on the chromosome
also show areas on chromosome with no genes (gene deserts) and areas where multiple genes are encoded and which directions they go
- hash marks -- repeated sequences (10 to 1000 base pairs long) also shown
- can also show enhancers and promoters
- ?

Distinguish between cis- and trans-regulatory factors (relative to some gene of interest).

- cis regulatory factors (enhancers) are regions of the DNA sequence which serve as binding sites for TFs
- trans regulatory elements or transcription factors are proteins bind to cis regulatory elements ad either serve as activators or repressors for transcription

Define "chromatin" and its composition. What are some differences between heterochromatin and euchromatin?

- chromatin is the components of chromosomes which includes proteins and DNA, complex found inn eukaryotic cells
- chromatin is 1/3 of each: DNA, histone proteins, and non histone proteins
- nucleosome is made of two copies of four histones and histone 1
-histone sequences are highly conserved
-euchromatin: open chromatin, more accessible to RNA polymerase ad transcription factors
-heterochromatin: closed chromatin, less accessible to RNA poly and TFs, unlikely to be transcribed, often gene poor and repeat rich, dark staining ad localized to periphery of the nucleus

What are the components and functions of nucleosomes?

-nucleosomes are made of two of each of core histones (H2A, H2B, H3, H4) and H1 and wrap around DNA, basic structural unit of chromatin
-fuctio to tightly pack DNA and change the state of chromatin based on modifications to them
-histone tails are exposed beyond the nucleosome and can have PTM by acetylation, methylation, phosphorylation, and ubiquitination of certain residues
- ex. histones can be acetylated so not tightly bound to DNA and then can be more open to be transcribed
- cann also cause altered recognition by cofactors which influences what other proteins will interact with histones, depends on residue at, depends what cause will be to transcription.

Histone modifications are essential for regulating chromatin state. What are major types of modifications and the enzymes that participate in them? What are their consequences for transcription?

-acetylation: histone acetyl transferase (HAT) acetylated histones on their Lys residue which makes them less attracted to DNA bc no longer charged nh3 group this makes them more open and are more readily transcribed, histone deacetylase (HDAC) removes the acetyl group from the lys residue which causes them to have the positive charged nh3 and are attracted to DNA, this means the DNA is held more tightly together and has less transcription
-methylation: protein arginine methyltransferase (PMRT) adds a 2' methyl to ARG, histone methyltransferase (HMT) adds 1, 2, or 3 methyl groups to LYS, consequences methylation not as predictable as acetylation

How can one infer the distribution of histone modifications (sometimes referred to as histone "marks") and non-histone proteins in chromatin using methods of second generation sequencing?

-ChIP-Seq: chromatin immunoprecipitation sequencing, chromatin with proteins is isolated from the whole genome, chromatin is fragmented into small pieces, chromatin is incubated with antibodies to the protein of interest, precipitate antibody-chromatin complexes (pull down certain antibody), sequence collected DNA fragments, align sequences to genome to detect regions enriched in the protein of interest
-differential enrichment of histone ' marks' correlated with transcriptional activity, higher peaks means that mod is more likely in that region of the DNA, more reads means more likely target protein was bound to the protein and histone has that modification

What traits do cancer cells typically evolve that are different from normal cells? Are there particular aspects of cancer cells that allow facilitate the evolution of increasingly malignant phenotypes?

traits of cancer cells:
- resist cell death
- sustain proliferative signaling
- evade growth suppressors
- activating invasion and metastasis
- avoid immune destruction
- deregulate cellular energies
- enabling replicative immortality
- inducing angiogenesis
exhibit grossly dysregulated gene expression: tumor suppressors down and oncogenes up

What evidence suggests an important role for H3K27 mutations in pediatric glioma?

-H3 mutations common in pediatric cancers (gliomas, chondroblastomas, giant cell tumors of bone)
-specific mutations associated with particular tumors types and locations
-diffuse intrinsic pontine glioma (PIPG), midline structures, median survival 9-12 months, H3 mutations in 80%
-H3K27 - LYS is usually mutated to a MET, MET cannot undergo normal LYS modifications (leads to dysregulation, genes normally repressed are now active)
-h3 mutation present in every of several cases of cancer in children

Distinguish ATP-dependent chromatin remodelers (e.g., SWI/SNF or chromodomain factors) from histone modifying enzymes (e.g., HDACs, HMTs). What are the shared features of ATP-dependent chromatin remodelers?

- ATP dependent remodelers work by nucleosome sliding, displacement and modification,
-ATP depended chromatin remodelers share: affinity for nucleosome, domains to recognize histone modifications, similar ATPase domains for overcoming nucleosome DNA interactions, domains for interactions with other proteins
- major families: SWI/SNF, CHD (chromodomian), ISWI
-ex. CHD1 large class proteins recruited to nucleosome can remodel chromatin in that region
- histone modifying enzymes often change affinity by changing charge (adding acetyl or methyl)
-ATP dependent remodelers can also associated with the nucleosome and DNA and ca expose sites (specific cis reg elements) for DNA binding proteins, can do this by site exposure (repositioning, ejection, or unwrapping), or by altered composition (can exchange dimers, histone variants, or eject dimers out of nucleosomes)

Describe the various ways that ATP-dependent chromatin remodelers can affect DNA binding site accessibility.

-modifers can not only change affinity but can also change positions
-ATP dependent remodelers can also associated with the nucleosome and DNA and ca expose sites (specific cis reg elements) for DNA binding proteins, can do this by site exposure (repositioning, ejection, or unwrapping), or by altered composition (can exchange dimers, histone variants, or eject dimers out of nucleosomes)

What is a "pioneer" transcription factor and what distinguishes it from other types of transcription factors? What kinds of interactions can pioneer TFs have, and in what contexts have they been shown to be especially important?

-DNA binding proteins that are able to bind target DNA sequences even in closed chromatinn
-can initiate chromatin remodeling, permit binding of other TF, histone variants, and chromatin remodelers
- stabilize open chromatin state
- play roles in cell programming and reprogramming (Stem cells)
-include FoxA, FOXO, GATA factors
go through three states:
silent state: chromatin scanning, initial targeting
competannt state: enable other factors to access
active state: cooperative stable binding w secondary TFs acquisition of active histone mods

Several DNA motifs in proximal promoters are essential for the commencement of transcription from particular genes. What are two of these motifs and how do they function to promote transcription?

-RNA poly 2 recruitment to promoters requires interactions with other proteins (general TFs)

How do "basal" or "general" transcription factors compare with tissue- or cell-type specific transcription factors?

-"general" transcription factors are involved in the formation of a PIC for the transcription process (ex. TFIIB (TATA binding protes), TFIIA -D -E -F -H
-cell type specific transcription factors will serve as either activators or repressors

What are the Pre-initiation and Mediator Complexes and how do they promote transcription? How do the proximal promoter and "distal" cis-regulatory elements (occupied or unoccupied) fit into the process?

- the PIC is a assembly of around 50 proteins in several different complexes (RNA pol, TFIIB, TFIIA, E,D,F,H), this TFs have to appear at the promoter to stabilize RNA, PIC has to form to recruit RNA pol to the promoter, construction of PIC is essential for RNA pol 11 to be recruited and stay on DNA to transcribe
-Mediator complex includes around 30 proteins subunits, promotes PIC assembly ad pol 11 localization, stabilizes PIC in vitro but short lived in vivo, integrates and communicates with TFs bound to cis regulatory (enhancer) elements, promotes looping of DNA causing spacially clustered activating TFs near the transcript start site, also regulated Pol 2 pausing after 30-60 nucleotides have been transcribed, mediator bridges PIC to cis regulatory sites more cis regulatory factors
-large complex formed between mediator and PIC gives the potential for many interactions with proteins to determine if transcribed
-due to similarity in promoter and enhancer sequences both can transcribe bi directionally, making eRNAS at the enhancer and some uaRNAs at the promoter

How does a single nucleotide mutation in a Duffy antigen gene confer resistance to malaria?

-Plasmodium parasite gets Into red blood cells by exploiting cell surface chemokine receptors "Duffy antigens"
- GATA transcription factors regulate the production of Duffy antigens by binding cis regulatory elements and acting as activators
-human resistance is due to a single mutation T-46C in the GATA DNA binding site, this reduces the ability of GATA to bind and reduces the transcription of the Duffy antigen/chemokine receptor (DARC) by 96%
-individua;s with the T to C change at the GATA bindinng site are resistant to malaria
- C mutation: much less transcription of the Duffy antigen RNA
-down to one base to determine if gene transcribed

Describe the possible steps by which a gene in a region of closed chromatin begins to be transcribed.

-gene can begin to be transcribed when histones are acetylated or changed to open up chromatin
- coactivators can have histone acetyl transferase or histone methyltransferase activity to promote nucleosome displacement and the opening of the chromatin, coactivators can also bridge activators to the sights of transcription
-activators can work in several ways: transcription (activator transcribed by one gene and then goes to bind to cis reg element and activate another), phosphorylation (activator is phosphorylated and becomes active to bind to cis regulatory element), co factor interactions (activator bound to another element but another interacts and binds replacing it), ligand binding (a hormone can bind to activator which activates it to bind to cis reg element), cleavage from inactive precursor (activator held in the cytoplasm by an inactive precursor and once detactched can go ad bind to cis reg element)

What are some of the general ways in which transcription factor activity can be regulated (for transcription factors acting as activators or repressors). Explain one example in which a single transcription factor can have dual roles.

- activators can be regulated by: transcription, phosphorylation, ligand binding, cofactor interactions, and cleavage from inactive precursors
- repressors can be regulated by: interacting directly with the PIC ad preventing binding, promoting closed chromatin by histone modification or recruiting chromatin closing enzymes, mechanisms to prevent transcription include: competition, quenching, cytoplasmic sequestration, and heterodimerization
- specific example of dual roles involves the T3 receptor: T3 binds to the TH receptors which converts them from repressors to activators for many genes, when T3 is bound other TFs bind to promote histone acetylation and opening of chromatin allowing the PIC and RNA pol 2 to bind and transcribe, when T3 is NOT bound repressors bind to the TH receptor site in DNA which lead to histone deacetylation and PIC and RNA POL2 cannot bind, so Genes are not transcribed

How does DNA become methylated and what are the downstream consequences of such methylation? Name a couple examples of methylation during development.

-CpG islands are near transcription factor binding sites where there is an abundance of G and C richness
-cytosine of the CpG dinucleotide can be methylated by DNA methyltransferase (DNMT)
-when the promotor is occupied by TFs this prevents the C from being methylated but unoccupied promoters can be targeted
- when TF are not bound DNMT ca methylated the C which prevents TFs from binding
-methylated Cs are recognized by methyl Cp-G binding proteins like HP1 which then drives histone methylation and chromatin condensation
-Hp-1 proteins form large complexes which shut down large sections of sequence to prevent transcription
- these are called epigenetic modifications: examples include hemoglobin switching, one form is methylated at one time so only other transcribed and then this changes and other form only made, another example is x-chromosome inactivation in which cats can have different marking depending on which X chromosome is shut down, methylation can be passed to daughter cells and in some cases offspring

Which strand of DNA is transcribed by RNA Pol II and in what direction? In what direction is the RNA molecule synthesized? What is a "transcription bubble" and over what region are RNA bases hydrogen bonded to DNA bases in an "RNA-DNA hybrid"?

-template strand is transcribed from the 3' to 5' direction to make an RNA which resembles the RNA-like or sense strand in the 5' to 3' direction, adds to the 3' end of the RNA being made
- the transcription bubble is the complex with RNA pol 11 where the two strands of DNA are melted apart and the RNA is being transcribed
-the RNA-DNA hybrid of bonding is 8 nucleotides long

What is RNA Pol II pausing and how is it regulated? Speculate on why pausing might occur.

- RNA pol 2 has early elongation called pioneer escape for 20-60 nucleotides and then undergoes pausing of pol 2
-by stopping this is another check to see if the gene is going to be transcribed
- pausing depends on interactions with the: mediator complex, NELF, DSIF, promoter elements, and stable RNA/DNA hybrids which might be hard to break (ex C-G 3 h bonds)
- proteins and elements of the promoter itself are involve in the pausing
- productive elongation then continues rapidly through termination
- pausing may keep genes POISED for expression and allow additional regulation and signal integration while maintaining access to the PIC

How has an ability to visualize single RNA molecules contributed to our understanding of transcription dynamics and the roles of core promoters and enhancers (cis-regulatory elements) in regulating the rate and intensity of transcription?

- single molecule RNA imaging has revealed discontinuous transcription
- allows us to track when mRNA is made
-transcription occurs in bursts
-the size of the burst depends on the promoter and how many RNA pol 2 are recruited at once
-the enhancers (cis regulatory elements) control the frequency of the bursts

What is the 5' cap on mRNA, how is it synthesized and what does it do?

- new transcript starts with a pppG or pppA but the mature mRNA has a methyl G-cap in reverse orientation added early in transcription
- the G is added by guanylyl-transferase
-the methyl is added by guanine-7-methyltransferase
-functions: protects transcript from degradation, recruits cap binding complex proteins which are important for transport from the nucleus, in the cytoplasm it recruits eukaryotic elongation factors (elF4G), RNA helices ad other proteins necessary for translation, it also interacts with the poly A binding protein to generate a pseudo-circular structure important for efficient translation

What are the basic events of mRNA splicing and how do specific sequence regions delineate where these events happen? How specific and predictable are these sequences?

- mRNA splicing typically occurs at the GU-AG splice sites with interrupting branch sites
- splice donor before a GT and splice acceptor after an AG typically, branch site is in the middle about 30 nucleotides long ad is usually CT and ACT
- sites are functionally equivalent across introns so correct splicing depends on the simultaneous recognition of corresponding GU-AG regions, if misses one AG or GT then could accidentally cut out Exon etc.
-spliceosome: small nuclear ribonucleoproteins (snRNPs) each comprise a small RNA with about 20 proteins
-four spliceosomes work together and this is done while RNA is still being transcribed
- cut at splice donor site and then branching occurs to the branch site and a 2' to 5' bond is made, creating a loop called a lariat
-then a cut is made at the splice acceptor and the two exons are put back together
- there is lots of room for error in splicing and misplacing can lead to disease
- may different variability and alternative splicing produces many different kinds of regulation of genens
- 20,000 protein coding genes to over 100,000 protein coding mRNAs
-variable features at core promoters and TSS (may have multiple promoters and which ones used determines which RNA is made), transcriptional termination or polyadenylation, also varies in splicing to retain diff exons ad introns
- alt splicing may retain (or not) certain exons or introns
-95-100 percent of mRNAs w more than one exon yield multiple mRNAs

Explain the roles for splice site enhancer/suppressor sequences and protein in determining whether splicing occurs at a particular site.

- cis regulators are the RNA motifs and are recognized by proteins that will determine whether expressed or not (ex. exonic/intronic splicing enhancers/suppressors, ESE)
- trans regulators are proteins that bind the RNA cis motifs: serine arginine rich proteins promote splice site usage and compete with repressive heterogeneous nuclear ribonucleoproteins (hnRNPs) and tissue specific regulators (these tend to repress splicing sites)
- this occurs AT THE LEVEL OF RNA, only recognize RNA after transcribed, diff combos of factors at different times leads to different splice forms coming out
- possible for introns to be retained Premature stop codon causes whole thing to be targeted for degradation

How might the speed of RNA pol II be regulated and what is a proposed mechanism by which this could result in alternative splicing to generate different mRNAs from a single gene?

-transcription rate differentially exposes exons to splicing factors
- histone methylation/acetylation influences RNA pol 2 speed
-faster transcription results in larger loops that are more vulnerable to being spliced out
- precise histone mod and chromatin state may favor some splice variants over others
- splicing and transcription occur simultaneously: when RNa pol 2 is moving really fast there is mix match between the speed of the splicesome and RNA pol 2 so larger loop that the spliceosome is associated with, sometimes this excess loop contains the exon and removes two introns and an exon instead of one intron, if moving slowly less likely to cut out exons
- in any given case cannot determined what specifically affects splicing, know some factors but not EXACTLy how splicing occurs

How common is alternative splicing and what are some examples of the phenomenon? What is an overall consequence of alternative splicing (e.g., in the context of the "one-gene / one enzyme" hypothesis) and how much do we know about the specific functions (if any) of most splice forms?

alternative splicing is persuasive
- 95-100% of pre-mRNAS with more than one exon yield multiple mRNAS (2 to thousands of variants per gene)
- splice acceptor and donor sites are invariant (how does cell know where to do alt splicing?)
- consequence of alt splicing is many mRNAs and proteins made from one pre-mRNA due to alternative splicing, one gene one enzyme hypothesis false
- we only know general factors affecting (cis and trans splicing regulators, transcriptional rate due to histone mods) but we do not know EXACTLY how splicing occurs

Is it possible to predict from sequence data alone the impact of a mutation on splicing or phenotype? Why or why not?

No, it is not possible to predict from sequence data alone the impact of a mutation on phenotype
- cannot determine what specifically affects splicing only know factors that affect it like (cis/trans splicing regulators, transcriptional rate)
- these factors occur only at the level of the mRNA and not the DNA sequence
- cannot be determined by the DNA sequence
- diff combos of factors at diff times leads to diff splice forms coming out

Can defects in alternative splicing lead to human pathologies? What is one example?

-significance of most normal splice variants and protein isoforms is NOT known
- titin is unique case:
largest protein known and regulates length and elasticity of striated muscle, alternative splicing between fetus and adults regulates cardiac muscle elasticity, depends on splicing factor RBM20 (RNA binding motif 20), this is a trans regulator of splicing, in fetus 2 splice forms of Titian have large middle region which keeps elasticity, in adults splicing switches so the protein lacks middle region, in mutant RBM20 you can't have this change in splicing as you become an adult and you will get fibrosis in cardiac tissue and cardiomyopathy, specific phenotype ONLY due to a change in the splicing pattern, mutant individuals can only make two elastic forms of larger titin. the gene that produces the RBM20 protein is defective

What is the origin of the poly(A) tail found on mature mRNA? Why is having a poly(A) tail important, and what proteins contribute to functional roles of the poly(A) tail?

-pre-mRNAS are polyadenylated at 3' end when transcription terminates
- RNA binding proteins associate with Pol 11 and recognize polyadenylation signals (AAUAA) in the 3' UTR, several present and choice is regulated
-RNA pol 11 transcribes the sequence and is recognized by factors including Poly A polymerase
-endonuclease binds and cuts at the G/U- rich target site 11-30 nt beyond the poly a signal
-poly a polymerase adds a poly A tail of 200-250 ATPs
-Functions poly A tail: binding sites for Poly (A) are RNA binding proteins in the nucleus (PABPN) and in the cytosol (PABPC)
PABPN: promotes Poly A polymerase activity (adds more A nucleotides), interacts w cleavage and polyadenylation factor to influence Poly A length and cut transcript, creates circular structure, functions in RNA export to cytosol
PABPC: interacts w eukaryotic initiation factors needed for ribosome function (elF4G, E), maintains loop structure critical bc protects from being chewed up by endonuclease (cannot access ends of RNA), promotes ribosome release (eRF3,1), blocks degradation of mRNA at 3' and 5' ends
-shorter poly A tail may limit opportunity for PABPC binding, translational efficiency and protection from nucleus

What explains the difference between the numbers of codons and the types of tRNAs found in eukaryotic cells?

- at least one tRNA for each AA but not necessarily all 61 corresonding to each sense codon
-due to wobble position this accommodates degeneracy w promiscuous pairing of standard and modified bases
- given Phe tRNA can recognize more than one Phe codon
- modified nucleotides can recognize other, example anticodon I can recognize codon U, C, A
-dont need 61 tRNA genes to recognize all 61 possible codons

What are the events of translation initiation, elongation and termination?

- initiation: mRNA has to associate with PABPC and elFs, small subunit recognizes the 5' methylated RNA cap and attaches to the 5' UTR, scans until it gets to the start codon (AUG) and then can accept the MET tRNA, small subunit charged with MET tRNA causes large subunit to come in and makes full structure, most proteins first Aa (AUG) sequence matters but also sequence around it called Kozak sequence (usually 3 US are AG, Us usually CG, and DS usually G) these are favorable for small ribosomes and without Kozak sequence not translated as efficiently, adds another level of control, elFs helps 40s join to 60s
- elongation: tRNA accepted into A site, links two AA detaching from the tRNA and moves into E site, one from A site moves to P site and E site leaves
- termination: release factor enters A site when hits the termination codon, causes ribosome to get off of the mRNA

What factors besides ribosomes are necessary for translation initiation?

1. 40s ribosome subunit must associate with eukaryotic initiation factors and MET tRNA, mRNA associated with PABPC and elFs
2. mRNA and 40s ribosome associate togehter
3. 40s subunit scans to get AUG start codon
4. elFs helps 60s join 40s
5 mature 80s starts translation

What are two examples of ribosomopathies and what are their genetic bases?

- Diamond Blackfan anemia: 4-5 cases per million live births, requires bone marrow transplant, RPS19, RPS24, RPS17 mutations impair 18s rRNA production which leads to less 40s ribosome until which leads to depletion of mature ribosomes, can also have RPL5, RPL11, RPL35A mutations leading to the depletion of the 60s subunit and mature ribosome, not enough functional ribosome so anemia because cells need vast amounts of hemoglobin and red and white blood cells have to constantly turn over if compromised in having to make enough protein then they are affected
- Treacher Collins Syndrome: mandibulofacial dystosis affecting 1 in 10,000 to 50,000 live births, problems w airway, swallowing , brain development, hearing, affected individuals are heterozygous for any of 120 mutations, TCOF1 (treacle ribosome biogenesis factor ab 93%) - treacle localizes to nucleolus where rRNas associate w ribosome proteins, interacts with ribosomal DNA and recruits RNA pol 1 to nucleolus, without treacle, RNA pol 1 no longer localizes to nucleolus and pol 1 not where it should be and not making rRNAs to patch into ribosomes, POLR1C or POLR1D (RNA pol 1 transcribe rRNA genes other than 5s rRNA, ab 6%), issue in this disease is that no enough ribosome because pol 1 is not localizing, often mutation only one allele because if both would have no functional ribosomes and not survive, range of severities depending on gene affected and nature of allele

Many (but not most) genes have mRNAs with upstream open reading frames in their 5' UTR. What is a commonly accepted function of these upstream ORFs and what observations support this idea? What kinds of genes seem to have these upstream ORFs

-uORFs reduce translational output for mRNAs that encode very potent proteins (growth factors), this reduction inn protein is normal and important for regulating differentiation in some contexts (nervous system)
-evidence: 5'UTR of PTCH1 hooked to fluorescent reporter gene, when 3 US ORFs were removed 5 fold more fluorescent protein was made, when 1 uORF mutated doubling in fluorescence intensity
- shows that uORFs in 5' UTR put the breaks on translation, makes translation LESS efficient
-often have uORFs for genes whose protein products are very potent (easy for too much protein to be made which is deleterious)
-with neuron cells: WT differentiate correctly into Foxa2, take one uORF that's missing and use PTCH coding sequence so much more PTCH protein made leads to much less differentiation to the FOXa2 state, too much PTCH1 poisons cells and doesn't let them differentiate
-why uORFs lower translational efficiency? multiple AUG means multiple places ribosome can bind and if there prevents other ribosomes from binding at the same time, where it recognizes the first AUG has a huge impact, multiple opportunities to start on small uORF and detach before moving protein, small majority would miss the stop and actually translate, evolutionary favorable to make small peptides than too much of a protein, uORF dont have good Kozak sequences recognized in some cases but also not in other cases

How do microRNAs contribute to regulating transcript and protein abundance?

- microRNAs: 21-24 nt noncoding miRNA guides bind to mRNAs, key regulators of mRNA abundance (60% of genes have miRNA target siteS), 1900 miRNA genes in human genome and each with multiple target mRNAs, miRNAs form an mi-RNa induced silencing complex (RISC) with the Argonaute protein, miRNA called a guide
-miRISC sometimes perfectly complementary to the coding sequence of an exon, binds and proteins in the RISC cut on either side, then the mRNA is chewed up by exonucleases
-also common miRNAs recognize mRNA even though not perfectly complementary, get bulge miRNA to align nucleotides in a few places, despite mismatching this binding efficient enoughjt to determine if translated, does this by blocking binding of formation of topology of mRNA to assemble ribosome (doesn't CUT but BLOCKS), blocks ELG and assembly ribosome
-phylogenetic variation: certain organisms do this process different ways, different organisms where miRNA are found, in vertebrates 5'UTR is often the target of miRNA, in others exons are targeted

Explain how cells remove transcripts that have premature termination codons or other kinds of damage that might result in defective proteins.

-mRNA ORF dictates the peptide sequence but nuclear export, localization in cytoplasm, engagement with translational machinery, create of degradation, etc depend on bound proteins (cap binding proteins, TIF, SR proteins, EJC)
- EJC core proteins and EJC interacting proteins regulate mRNA function, each EJC has four proteins and EJC is loaded onto RNA as splicing occurs and follows the spliceosome, occurs every 24 nucleotides US of each exon-exon junction, cover open reading Frame but absent from 3' UTR, as splicing continues one EJC is added per each exon exon junction, this recruits other proteins to the RNA (EJC interacting proteins) which influence remaining splicing, facilitate export from nucleus, promote initial translation, and are required for quality control
- non sense mediated RNA decay: normal transcript has EJC and assessors proteins, as transcript translated 1st ribosome moves down and pops off the EJC, once at end of transcript no EJC left, if there is a PTC upstream of a EJC site than the 1st ribosome will stop at this PTC and detach from the mRNA, if EJC is still one then more proteins are recruited which target for degradation
-WHERE STOP CODON AND WHERE LAST EXON EXON JUNCTION IS ARE CRUCIAL
-NMD discovered in B-thalessemia (premature stop codons cause transcript loss and reduced hemeglobin)
-viruses interfere with NMD mechanisms to promote their own replication inc HIV, HEP C, ZIKA, virus can interfere with assembly degradation complex or assembly of EJC, makes the process fail so their own RNA can code for multiple proteins on one RNA without being degraded

mRNA abundance is relatively easy to measure and this can even be done at large scale by single cell RNA-sequencing, in which transcripts for thousands of genes are isolated and counted for every cell separately from a tissue or embryo. By contrast, protein quantification is much more challenging, and especially so at single-cell level. What are the risks of inferring gene function from mRNA abundance data alone? How predictive are such data for estimating protein abundance and why?

-gene expression and activites of gene products are regulated at many levels: abundance at one level does not often predict abundance or activity at another level
- Many steps where can have regulation from gene to protein: genome (whether gene expressed), transcriptome (bursts transcription, splicing, transport, volume control how long transcripts persist due to Poly A and miRNA), translation (how efficient and how long), proteome (folding and PTM)
- risks bc mRNA may be changed in various ways from its initial point as a gene due to splicing, Bursts transcription, Poly A, miRNAs etc.)
- predicting abundance of a protein based on abundance of mRNA is NOT easy (NOT 1 to 1), many places in between where mods can occur, mRNA is a poor predictor of protein content

eIF

eukaryotic initiation factor
-associates with mRNA and PABPC
-associates with 40s ribosome subunit
-helps join 60s subunit to 40s subunit

miRNA

-a class of functional RNA that regulates the amount of protein produced by a eukaryotic gene
-microRNAS
- key regulators of mRNA abundance
-1900 miRNA genes in human genome each multiple target mRNAs
-forms miRISC can target mRNAs for degradation or block translation

pre-miRNA

made as primary miRNA transcript, NOT protein coding but has exons and introns,
goes through drosha to clip hairpin, then to cytoplasm, then dicer clips bottom and forms an RISC

ribosomopathy

diseases caused by abnormalities in the structure or function of ribosomal component proteins or rRNA genes

upstream ORF

reduce the translational output of mRNAs coding for very potent proteins

reporter gene

Gene encoding a protein whose activity is easy to monitor experimentally; used to study the expression pattern of a target gene or the localization of its protein product, used fluorescent reporter genes to study uORFs in PATCH 5'UTR

RPL/RPS genes

RPL - genes coding for large ribosome subunit proteins
RPS - genes coding for small ribosome subunit proteins

40S/60S/80S ribosome/subunits

40S - small
60 S - large
80S - entire ribosome unit

EJC

Exon junction complex proteins, deposited on mRNA after spliceosome as splicing occurs,
knocked off by ribosome during translation to ensure no PTC

NMD

Nonsense-Mediated RNA Decay
surveillance system in eukaryotes that recognizes and eliinates mRNA's encoding nonsense codons with their protein coding regions, recognizes EJC still present after ribosome is knocked off at a PTC

What are the cellular and phenotypic consequences of prenatal Zika virus infection?

- virus affects the neural stem cells in the brain, impacts the ability to undergo normal mitosis, DNA may be replicated but not aliquoted correctly to daughter cells, chromosome segregation defects, sometimes multiple copies of one chromosome and when incorrect chromosome complement cell undergoes death and nucleus breaks down
- phenotypic consequences: severe microcephaly, decreased brain tissue and specific brain damage, seizures, auditory defects, scarring at back of eye, limited mobility, infects neural system cells

How do growth factors promote the G1 to S transition in normal cells?

-cell cycle reentry depends on growth factors like ligands that activate the receptor tyrosine kinase pathway, ligand is form of a dimer and receptors are dimers too, once bound transphosphorylate each other and go through a series of phosphorylations to activate transcription factors including cyclins
- cell cycle progression requires cyclins and cyclin dependent kinases (CDKs)
- when the cyclin-CDK complex forms phosphorylation occurs, many target genes to transition one phase of cell cycle to next
-about 10 cyclins that allow CDKs to function
-about 20 CDKs each with conserved structures
-CDK-cyclin complex phosphorylated hundreds of target proteins at Ser/Thr residues
-phosphorylation enable specific steps of the cell cycle
- example: RTK signaling drives transcription of cyclin D and E, form CDK4-cyclinD and CDK2-cyclin E complexes, these phosphorylate Rb which is bound to E2F, E2F is inhibited when bound to Rb but when Rb phosphorylated is released, E2F is transcription factor and promotes expression off genes needed for DNA replication
-Rb product of RB first identified as tumor suppressor, when Rb un functional E2F replicates too much leading to cancers

How do p53 and CDK inhibitors like p21 prevent cells from entering S phase when they have extensive DNA damage?

- p53 is transcription factor that is one of 'quality control' checkpoints
- p53 prevents S phase by inducing CDK inhibitors like p21, p21 inhibits activity of CyclinD-CDK4 and CyclinE-CDK2 complexes, can no longer phosphorylate Rb so it is bound to E2F and E2F cannot promote transcription of genes needed for DNA replication
-p53 also able to initiate programmed cell death when damage severe
-mutations p53 lead to cancer suseptibility, most cancers have p53 mutations

What are "tumor suppressors" and what kinds of roles do they play during normal development or homeostasis?

-tumor suppressor genes code proteins that regulate cell division
-example is RB (identified as tumor suppressor) which is the gene that codes for Rb, Rb binds to E2F (a transcription factor that promotes transcription genes related to DNA replication), Rb inhibits E2F, negative control
- example is p53, transcription factor that can induce cell death when damaged, can also induce p21 which inhibits CDK-cyclins from phosphorylating Rb

How does tumor suppressor copy number relate to cancer susceptibility in humans and other organisms?

- long lived animals, enormous amount of cells and larger lifespans, more room for mutations and errors in genes like p53
- would think have higher risk of cancer
- but they DONT have higher risk
- this is because have multiple duplicates of p53
-elephants have 20 copies of p53
- large bodies long lived species have compensated over time by evolution by duplicating the tumor suppressor genes

What experimental observations first supported a semi-conservative mode of DNA replication? How might these findings have differed were another mode involved?

-Meselson and Stahl
- used e-coli grown in two different mediums (14N and 15N)
- grew e-coli in 15N so that DNA contained heavier nitrogen then put this into a 14N, after one round of replication there was one strand 15N14N (in between the two weights of 15 and 14) after two round of replication there were two strands one light 14N14N and one medium 15N14N, this supported semiconservative because the two separated and each served as a template for the next replication, after each round replication DNA has one strand old and one strand of newly synthesized DNA
- if this were conservative, the 1st generation would have shown one 15N band (heavy) and one 14N band (light) because after one round one strand would be identical to the parent, so one DNA molecule would be all heavy and one all light
-if this were dispersive the first generation would be a middle weight of 15N14N as recorded but the second generation would also have one line at 15N14N because the DNA would be interspersed still, the old and new DNA would be interspersed in the daughters

What proteins function in DNA replication besides DNA polymerase and what are their activities?

-at potential origins of replication the pre replication complex (preRC) forms containing heterohexamer origin recognition complex (ORC), mini chromosome maintenance proteins, cell division cycle six protein, preRCs license potential origins for use
-helicase (mini chromosome maintenance proteins): unwind the DNA ONLY in multicellular organisms, separate the strands of DNA
- single strand binding proteins (replicating protein A): bind to the single strands and keep them in their open configuration
- primase (pol a): adds the RNA primer needed by pol delta to replicate the lagging strand, primers have to be Made along open strands of DNA (are actually RNA primers, short oligotides of RNA placed on DNA), RNA is chewed out later by exonucleases
-sliding clamp (PCNA): makes sure the new strand and the template stick together
-DNA polymerases: pol epsilon for the leading strand and pol delta for lagging strand

What are origins of replication and what does it mean for one to be "licensed"? Are there particular characteristics that make a site likely to be licensed? Are all sites that are licensed actually used?

-30,000 to 50,000 origins activated per cell division
- origins recognized by 12 bp autonomous replication sequence in yeast, AT rich islands in another yeast but in metazoans is unknown (maybe CPG islands and promoter sites?), may depend on higher topology of the chromosome
- assembly at the potential origins of a pre-replication complex (pre-RC) with herterohexamer origin recognition complex (ORC), mini chromosome maintenance proteins, and cell division cycle 6 protein
- PreRCs lisence origins for use make them potential site where replication could commence but not all origins licensed are used
-constitutive replication sites - origin used in every single cell
- inactive or dormant site - origin need used in any cell, DNA rep never starts at that site
- flexible cluster - site may get used or not depending on cell (vary depending non ells types)
- unknown why some not used

In what direction is the DNA template read and in what direction is a new strand synthesized?

- DNA template is read from 3' to 5 ' and new strands synthesized from 5' to 3'
- DNA pol can only add to the 3' end of the growing strand

Distinguish between lagging and leading strand replication and the proteins required for them.

-leading strand: strand that moves from 3' to 5' of template, one RNA primer can be laid down at 5' end of new strand and then DNA pol e can synthesize continuously, requires MCM (helicase), DNA pol e, and PCNA (sliding clamp), also RNA primer has to be chewed up later by exonucleases
-lagging strand: strand that moves from 5' of 3' end of template, cannot be synthesized continuously, instead forms short segments called Okazaki fragments where new RNA primer is laid down and then created new DNA from 5' to 3', DNA synthesized inn opposite direction of fork, requires MCM, DNA pol delta, PCNA (sliding clamp), pol a primate (for RNA primers), ligase to hold the separate fragments together, another DNA polymerase to fill inn gap after exonuclease chew out RNA

What are the consequences of DNA replication for chromatin state?

- old nucleosomes removed and histones dissociated, after replication have to reform
- euchromatin modifications not maintained
- heterochromatin modifications are maintained because these regions have no active transcription occurring, enzymes can regonize the presence of these modifications and add them to the new nucleosomes called "read-write" methylation, this is how epigenetic inheritance occurs, highly silenced regions remain silenced in daughters
- doubling DNA has to double histones and nucleosomes and proteins (is rep conservative all 8 stay or semiconservative 4 and 4?), histones do come apart to certain extent in replication some histones tend to stay together when nucleosome broken apart and some do not