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nuclear envelope
two lipid bilayers (inner and out nuclear envelope)
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Interstitial space (define and say what it is connected to)
lumen of nuclear envelope; continguous with the lumen of the endoplasmic reticulum
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nucleoplasm
- fluid phase interior of nucleus
- contiguous with cytoplasm but there is no free flow
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nuclear strucutre
conferred by a system of proteins in and out of the nucleus
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KASH
integral membrane protein that spans the outer envelope of the nucleus
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SUN
integral membrane protein that spans the inner envelope of the nucleus
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intermediate filaments
cytoskeletal molecules that are not A/GTPases
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nuclear lamins
- intermediate filament proteins that live just inside the surface of the inner nuclear envelope
- phosphorylation induces disassembly
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Histones (list, charge, pH)
- family of proteins: H1 [H2A,H2B,H3,H4]
- positive charge
- basic pH
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nucleosome
- DNA looped around histone complex (10nm)
- connected by linker sequences
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solenoid (define and what holds it together)
- coil wound into a tightly packed helix
- histone H1 holds it together
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chromatin
combination of DNA and the proteins associated with it (eg histones)
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heterochromatin
- very dark, dense, and very protein rich; tighly packed
- generally not unraveled
- found at telomeres and centromeres
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euchromatin
- lighter/less dense than heterochromatin both in the amount of DNA present and the amount of protein present
- partially unwrapped chromosomes
- makes the genes for proteins to transcribe them (transcriptionally active)
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aceylation
adding an acetyl group to a histone to neutralize some of its positive charge to allow thte histones to release the DNA partially so it can unravel
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semiconservative replication
conserve half of the genetic material
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Licensing/priming
When?
What?
Why?
- occurs during G1
- building up components around the origin for replication
- limites replication to a single cycle by building up the martials that will be used for replication
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ORI
- origin
- a DNA sequence that is A-T rich (easier to separate than C-G)
- 1000s in eukaryotes are scattered throughout chromosomes
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ORC
- origin recognition complex
- pre-replication complex; structural molecule
- six proteins (ORC1-ORC6)
- binds origin; serves as a landmark
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Cdc6p
- pre-replication complex; structural molecule
- protein that binds to the ORC complex and Cdt1
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Cdt1
- Cyclin dependent transcript #1
- pre-replication complex; structural molecule
- binds Cdc6p and MCM
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MCM
- mini-chromosome-maintenance
- heterohexomer (MCM 2-7)
- provide helicase activity to separate DNA strands
- activated by Cdc45p
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Cdc45p
binds MCM complex and activates it
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RPA
- Replication Protein A
- binds single stranded DNA (ssDNA) and stabilizes it in its single stranded form
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DNA dependent DNA polymerases
phosphodrestal bond: bond with 3' hydroxyl of one base and the 5' phosphate of another base
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DNA polymerase a(lpha)
- contains primase activity
- first few bases are RNA primary followed by DNA primer bases
- error prone
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RFC
- Replication Factor C
- recognizes primer and binds to it
- binds to PCNA
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PCNA
- Proliferating Cell Nuclear Antigen
- binds RFC; wraps around DNA
- binds DNA polymerase d(elta)
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DNA polymerase d(elta)
- longer runs
- requires a primer
- proofreading activity
- low error rate (1/10,000,000)
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DNA ligase
seals the gaps in DNA
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Toporsomerase I
cuts a nick in one strand of DNA to unwind strands (relieve stress)
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Toporsomerase II
cuts two strands and ties them back together (requires ATP)
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What happens in early G1 stage?
- ORC is bound to the origin
- Cdc6 and Cdt1 bind
- recruit MCM (but don't turn it on)
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Early S-Phase (part 1)
- cdc45p binds MCM complex and activates its helicase activity
- replication bubble is opened p
- ORC, cdc6 and cdt1 leave the complex
- ORC returns at the end of S-phase; cdc6 and cdt1 do not return until G1
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Early S-Phase (part 2)
- replication forks start to extend
- RPA is recruited (keeps strands separate)
- DNA polymerase a(lpha) is recruited
- primase synthesizes primer (RNA - DNA) reading 5'-3'
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Early S-Phase (Part 3)
- RFC is recurited by primer and is bound to it
- RFC recruits PCNA
- PCNA recruits DNA polymerase d(elta)
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RNA portion of primer in replication is removed by ____
RNAase H
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what limits the amount of times a cell can replicate its DNA?
shortening of telomeres
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What is a difference between RNA and DNA?
the presence/absence of hydroyl on C2 ribo/deoxynucelic acid
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RNA
- shorter sequences
- base pairs w/ it self creating hairpin loops (short) and stem looks (large)
- tertiary structure in some RNAs
- can be catalytic
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ribozymes
splicing of mRNA catalyzed by RNA
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Transcription:
Read:
Write:
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Differences between replication and transcription
- 1. entire genome is not transcribed
- 2. Takes place throughout most of the cell cycle
- 3. primers are not required
- 4. RNA polymerases are much less accurate than DNA polymerases (b/c molecules are disposable)
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RNA Polymerase I
makes rRNAs
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RNA Polymerase II
mkaes mRNAs
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RNA Polymerase III
makes tRNA and snRNA (small nuclear RNA)
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promoter (transcription)
- just upstream from coding sequence
- transcriptional machinery binds here
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TATA box
- 7-9bps long
- specific binding site and recognition site for transcription factors
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core promoter
- inside promoter
- builds transcriptional machinery
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Regulatory Sequences
upstream from the gene. Serves as a recruting element for transcription factors
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Transcription facors
proteins bonding DNA-> recruiting/activating polymerase
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General transcription factors
- required for transcription of all genes
- bind in promoter region (TATA box)
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promoter-proximal elements
DNA sequences of transcriptional start site
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TFII-D
- transcriptional factor
- TBP-TATA box binding protein
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TFII-A,B,F
- transcriptional factor
- bind to each other, the TATA box, and PolII(makes mRNAs)
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TFII-E
- transcriptional factor
- binds TFII-H and PolII(makes mRNAs)
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TFII-H
- binds to TFII-E and Pol II
- binds helicase and kinase(phosphorylated PolII)
- opens strands + transcription bubble
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Polymerase Pol II
- makes mRNA
- needs to be phosphorylated before it can be activated
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CAC
- capping enzyme complex
- binds to the 5' base of mRNA
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5'cap (3)
- stabilizes mRNA
- serves as a signal for nuclear export
- signals translation of gene
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splicing (what is it and what is used to do it)
- occurs after transcription with a spliceosom (composed of RNA and proteins)
- cuts out introns and ligates exons back together
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What happens during termination of transcription?
- 1. Polyadenylation signal (AATAAA) signals polymerase II to slow down and stop
- 2. Termination facts bind polymerase II and mRNA to cleave the mRNA
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polyadenylation
- Poly A polymerase binds 3' end of mRNA and adds 100-300 As (polyAtail)
- serves as a timer- when too many As have fallen off, the mRNA is enzmatically destroyed
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translation error rate
1/1000 amino acids
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where does tranlation take place?
cytoplasm in ribosomes (mRNA must be translocated through nuclear pores out to the cytoplasm)
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mRNA is read __ to __ in translation
amino acids chain is synthesized ___ to ___
- 5'-3'
- N-terminal to C-terminal
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when in the cell cycle does translation take place?
whenever
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start codon for translation (and what it codes for)
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A
P
E
translation sites
- A(dd): new tRNA enters ribsome here
- P(olypeptide): contains tRNA that is linked to a growing polypeptide chain
- E(xit): bound to tRNA that has lost its amino acid
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tRNAs have a ____ structure
cloverleaf
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what code do tRNAs end with a the 3' end? what is attached at the 3' end?
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MIFs
- mRNA Initiation Factors
- bind 5' cap and recruit small ribosmal subunit
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eIF2
- Eukaryotic Initation Factor 2
- when turned on (bound to GTP) it prevents translation until the GTP is hydrolysized to GDP (when AUG is reached) (allows large ribosomal subunit to bind)
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g-protein
- small proteins that serve as switches
- bound to GTP (on) until hydrolysis is trigged externally (GDP-off)
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EF1
- elongation factor 1
- it is an activated g-protein that prevents peptide bond formation unless the codon and anticodon match (GTP is hydrolyzed when there is a match)
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EF2
- Elongation Factor 2
- an activated g-protein that ensures that peptide bond formation is physically coupled to ribosome translocation in the 3' (->) direction down mRNA
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what catalyzes the peptide bond between amino acids in translation? what happens after that?
- large ribosomal subunit
- EF2 hydrolyzes GTP allowing ribosome to move forward 3 bases (1 step)
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how does translation end?
- stop codon is reached
- termination factor enters A site
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signal
primary piece of information received by the cell
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3 types of signals
- hormones (chemical)
- damage (physical)
- cell to cell contact (physical)
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signal transduction system
- the component that receives and responds to the signal
- can be a single cell, group of cells, tissue, organism
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4 types of cell signaling
- 1. endocrine
- 2. paracrine
- 3. juxtacrine
- 4. autocrine
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endocrine signaling
- longest range
- chemical (eg hormone)
- source: gland
- gets to target through blood
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paracrine signaling
short ranges (less than a mm)
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juxtacrine
- very short rainge (direct cell to cell contact)
- signal produced on surface of cell
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autocrine signaling
- cell signals to itself
- signal released is received by a receptor in its own plasma membrane
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general mechanism of signal transduction (5)
- 1. receptor-ligand bonding
- 2. phosphorylation
- 3. use of g-proteins
- 4. 2nd messengers
- 5. localization
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phosphorylation
addition of a phosphate group (rapid, catalytic)
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kinsase
- enzyme that phosphorylates proteins
- many; protein specific
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phosphatases
- proteins that remove phosphates
- low in number, less protein specific than kinases
- always active unless turned off
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Familes of kinases
- 1. serine-threonine kinases (S/T) (most common)
- 2. tyrosine kinsase (Y)
(a few kinases also have duality)
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GEFs
- Guanine nucleotide Exchange Factor
- knocks off GDP from g-protein and allows GTP to attach
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GAP
- GTPase Activating Protein
- binds to GTP-bound g-protein and causes it to hydrolyze GTP to GDP
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examples of second messengers (4)
- ions (Ca2+)
- lipids (PIP2)
- carbohydrates (IP3)
- nucleotides (CAMP)
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receptors are ________ proteins (name domains)
- tripartite
- n-terminal, middle, c-terminal
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n-terminal of receptor
- transcriptionally active domain (TAD)
- recruiting factor for activators of transcription
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middle domain of receptor
DNA binding domain
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