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316 Cards in this Set
- Front
- Back
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Genes encode:
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1. mRNA
2. structural RNAs 3. bioactive RNA |
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mRNA
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messenger RNA that encode proteins
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gene
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entire nucleic acid sequence that is necessary for the synthesis of a functional gene product
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genome
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total genetic material possessed by an organism
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human genome size
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3,000,000 kb
25,000 genes |
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bacteria genome size
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4700 kb
4405 genes |
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base pair of DNA
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0.34 nm
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1 turn of DNA
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10 base pairs
3.4 nm |
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length of human chromosomes
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17-85 mm long
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spaghetti analogy
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-spaghetti is one million times larger than DNA
-if we make the chromosome the size of spaghetti, it will be 85 km -53 miles: from Athens to Atlanta |
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Why is DNA packed into the nucleus?
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-to protect it from breakage
-to arrange into an ordered structure (no tangles) -to separate RNA synthesis (transcription in nucleus) and protein synthesis (translation in cytoplasm) |
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chromatin
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DNA + histones + non-histone chromosomal proteins
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What are the most abundant proteins in chromatin?
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histones
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Non-histone chromosomal proteins
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1. transcription factors
2. high mobility group (HMG) proteins 3. scaffold associated proteins |
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HMG proteins
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essential proteins required for transcription that are thought to work by remodeling chromatin and facilitating transcription factor binding
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Yeasts that lack HMGs....
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are not viable and have defects in transcription
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What does chromatin in low salt look like?
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beads on a string
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What does chromatin in physiological salt look like?
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30nm chromatin fiber
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Is chromatin found in prokaryotes?
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No
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Beads on a string is associated with...
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euchromatin
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30nm fiber is associated with...
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heterochromatin
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Four Types of Histones:
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H2A, H2B, H3, H4
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What allows assembly of nucleosomes?
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histone fold
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Which histones form dimers?
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H2A and H2B form two dimers and H3 and H4 form a tetramer; these assemble to form a histone octamer
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What is the importance of the histone dimers?
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stabilizing the molecule
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What happens when the histone octamer dissociates from DNA?
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It falls apart into the H3/H4 tetramer and two H2A/H2B dimers
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How do the histone dimers and tetramers form?
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Through the histone fold
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How do histones like to bind to DNA?
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With two AA/TT sequences spaced 10 bps apart (minor groove inside)
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Why do histones bind to AA/TT sequences rather than G/C sequences?
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The DNA needs to compress to bind to the nucleosome and the AA/TT sequences facilitate this better than G/C
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What is the role of nucleosome-positioning cues (AA/TT) in transcripton and chromosome structure?
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Unknown
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What does the nucleosome consist of?
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histone octamer + DNA
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What mediates histone packing?
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Histone H1, which can bind to additional 20 bps
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What are the two different 30nm chromatin structures?
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1. solenoid
2. zigzag |
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solenoid versus zigzag
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solenoid based on structure of longer nucleosomes while zigaag based on structure of 4 nucleosomes
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How can you remove histones from chromosomes?
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treatment with liidosalicylate detergent
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Why are chromosome loops visible in lampbrush chromosomes?
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due to extensive transcription occurring
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What is a lampbrush chromosome?
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largest chromosome known and is found in amphibian oocytes
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What makes chromosome loops visible on meiotic chromosomes?
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hnRNP particles
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condensins
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protein complexes that use ATP-hydrolysis to condense mitotic chromosomes
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Structure of condensins
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-two SMC dimers that can hydrolyze ATP
-other proteins |
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How are condensins activated?
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By phosphorylation by mitotic kinases
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SMC proteins stands for...
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structural maintenance of chromosomes
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What SMC proteins are found in condensins?
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SMC2 & SMC4
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What contains SMC1 and SMC3?
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Cohesins + cap proteins to bind sister chromatids
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How does condensin function?
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1. The hinge region of SMCs bind to DNA and initiates ATP hydrolysis of the head region
2. This separates the bound head regions 3. The hinge region continues to bind DNA, while the head region can rebind ATP and form a dimer with another head region 4. This traps DNA |
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Basic Function of Condensins
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1. Release
2. Sitting 3. Hooking 4. Trapping/Gathering 5. Condensation/segregation |
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Regions of chromatin that are being actively transcribed have regions of:
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1. Nucleosome-free DNA
2. lack histone H1 3. acetylation of histones H3 and H4 on specific lysine residues |
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Acetylation _______ DNA-nucleosome connection
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weakens
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histone acetyltransferases
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enzymes that acetylate conserved lysine amino acids on histone proteins by transferring an acetyl group from acetyl Co-A
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What suggests that acetylation is essential?
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mutation of yeast histone H4 acetylated lysines to arginine is lethal; both amino acids are + charged, however arginine cannot be acetylated
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Are histone H3 and H4 acetylated in mitotic chromosomes?
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No
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How does acetylation work?
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histones are + charged and DNA is - charged, so they attract and lock together; actylation breaks the charge and loosens the histone arms from the DNA, allowing transcription to occur
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Acetylation of histones ______ transcription and deacetylation ______ transcription
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facilitates; represses
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How can lysine residues in histone tails be modified?
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by acetylation or mono-, di-, or tri-methylation
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What other ways can histone tails be modified?
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phosphorylation of serine amino acids or mono-ubiquitination of lysine residues
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methylation of K-9
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heterochromatin formation and gene silencing
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methylation of K-4 and acetylation of K-9
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gene expression
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phosphorylation of S-10 and acetylation of K-14
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gene expression
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methylation of K-27
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silencing of Hox genes and X chromosome inactivation
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Function of H3.3 variant
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transcriptional activation
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Function of CENP-A variant
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centromere function and kinetochore assembly
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Function of H2AX variant
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DNA repair and recombination
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Function of H2AZ variant
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gene expression, chromosome segregation
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Function of macroH2A variant
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transcriptional repression, X-chromosome inactivation
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In some cases, what can happen if histone variants are present prior
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propagation through S phase
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Does the DNA around nucleosomes always stay closely wound?
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No, sometimes unwind, allowing a DNA-binding protein to bind
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What do chromatin remodeling complexes do?
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Shift nucleosomes to altered positioning or remodeled nucleosomes and facilitate transcription
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How do chromatin remodeling complexes facilitate transcription?
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1. Remodeling complex A binds, loosening the DNA so that DNA-binding proteins can bind
2. Gene expression, DNA replication, and other processes that require access to DNA packaged in nucleosomes can occur 3. Remodeling complex B binds and DNA-binding proteins dissociate 4. Standard nucleosomes are restored |
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What are some chromatin remodeling factors and what do they do?
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HP1, PCC, SWI/SNF; they shift chromatin into and out of the 30nm fiber
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Heterochromatin is ________ to DNAse I and euchromatin is ________ to it
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resistant; sensitive
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human karyotype
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consists of 46 chromosomes
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Giemsa stain
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historically used to distinguish chromosomes
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FISH
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-fluorescence in situ hybridization
-DNA corresponding to each chromosome is labeled with a unique fluorescent dye -mitoitc chromosomes are denatured and hybridized to the probes -UV light of defined wavelength is used to excite the fluorescence of the dyes to label each chromosome |
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What can FISH be used for?
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to map chromosome changes through evolution
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How do the base pairs match up in DNA replication?
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C and G with 3 hydrogen bonds; A and T with 2 hydrogen bonds
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Why are high energy bonds needed?
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because the phosphates have a high number of negative charge that repel each other and cause problems
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How many phosphates are released in DNA replication?
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2
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What are the problems that must be solved for DNA replication?
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1. DNA polymerases are unable to unwind DNA to separate the two strands
2. DNA polymerases can only catalyze nucleotide addition to the 3' end and DNA is antiparallel 3. DNA polymerases only elongate an existing 3' extension of DNA or RNA |
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helicases
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separate DNA strands while hydrolyzing ATP
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What is the structure of a helicase?
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six subunits that each hydrolyze ATP; subunits can be different protiens
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What does synthesis in the lagging strand involve?
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many Okazaki fragments that are composed of RNA primers and then the synthesized DNA
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How long are the RNA primers in eukaryotes?
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around 10 nucleotides long and spaced 100-200 nucleotides apart
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How does DNA ligase work?
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-uses ATP to join the nicks
-2 phosphates of ATP are hydrolyzed to link AMP to the 5' phosphate of the DNA strand, producing a linked ADP -the linked ADP is hydrolyzed to AMP during the joining of the 3'OH group to the 5' phosphate of the DNA backbone |
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polymerase ______ replaces polymerase ______ after the RNA primer to reduce error in replication
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delta; alpha
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PCNA
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-proliferating cell nuclear antigen
-increases processivity of DNA polymerase delta -clamp together around DNA strand and RFC clamp loader |
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Does the clamp loader stay on the DNA strand the whole time in the case of PCNA?
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No, it falls off; just used to load PCNA onto DNA strand
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What keeps the unwound strands from winding back up?
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single strand binding proteins
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RPA
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-rep protein A
-binds to DNA template with short region of base-paried hairpins and introduces monomers that straighten the DNA region so that it doesn't form secondary structures |
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TAntigen in SV40 system
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recognize and open origin (helicase) to allow primosome loading
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RPA in SV40 system
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ssDNA binding facilitates helicase loading
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Pol-alpha primase in SV40
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RNA-DNA primer synthesis
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RFC (replication factor C) in SV40
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binds primer and facilitates PCNA binding
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PCNA in SV40
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ring-clamp for polymerase
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FEN1/MF1 in SV40
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nuclease to remove RNA primers
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Pol-delta in SV40
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leading and lagging strand synthesis with 3' to 5' exonuclease activity
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RNase H1 in SV40
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nuclease to remove RNA primers
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DNA Ligase 1
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joins Okazaki fragments
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Topoisomerase I
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relieves helical tension
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Topoisomerase II
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untangles DNA
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Initiation of SV40 replication
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1. T antigen binds at origin
2. DNA unwound and RFA binds to template strand 3. Primase pol-alpha binds and primer synthesis begins 4. RFC binds and DNA chain synthesis by pol-alpha is stimulated by the RFC 5. PCNA inhibits elongation and displaces primase pol-A 6. binding of pol-delta to PCNA/RFC leads to the synthesis of leading strands while binding of primase pol-alpha and RFC to the lagging strand template leads to synthesis of the lagging strand discontinuously |
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pol-alpha
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functions in a complex with primase to initiate DNA synthesis immediately after RNA primer
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pol-delta
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completes both lagging strand and leading strand synthesis for SV40 DNA; for genomic DNA only lagging
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pol-epsilon
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completes leading strand synthesis for genomic DNA
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pol-beta
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functions in DNA repair
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pol-gamma
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functions to replicate mitochondrial DNA
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Which polymerases have 3' to 5' exonuclease activity?
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pol-delta, pol-epsilon, pol-gamma
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polymerase
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enzyme that facilitates the polymerization and repair of DNA or RNA
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What is the proposed mechanism for removal of an RNA primer not in vivo?
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-using RNase H1 and FEN1
-RNase H1 cannot remove RNA-DNA bond, so removes primer except for last nucleotide -FEN1 binds and cuts off the last nucleotide |
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What is the likely in vivo mechanism for removing an RNA primer?
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-DNA primer displaced by Dna2 helicase
-flap removed by FEN1 -removes DNA primer that does not have proofreading -pol-delta can also generate flap by strand displacement, but dna2 helicase still needed |
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How often is an error made in replication?
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once every 10^9 bps
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Tautomeric nucleotides
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-special forms of a nucleotide that can incorrectly base pair
-tautomers shift back to normal which destroys the base pair -base is removed by DNA pol |
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Topoisomerase I
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-has tyrosine at active site and covalently binds to a DNA phosphate
-this breaks the phosphodiester linkage in one DNA strand -the two ends of the double helix are able to rotate, which relieves accumulated stress -the original bond energy is stored in the phophotyrosine linkage, making it reversible -the phosphodiester bond spontaneously reforms, regenerating both the helix and the topoisomerase |
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Topoisomerase II
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-after replication, different helices tangle
-topo covalently attaches to both interlocked strands of DNA helices, which interrupts the helix of one strand and forms a protein gate -the gate opesn and shuts, letting the second dNA helix pass through -covalent bond reversed and restores intact double helix -in beginning, ATP binds to dimerize the domains of topo |
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Which topoisomerase does bacteria use?
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topo II only
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Topo II is also known as...
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gyrase
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What stops Topo II from further tangling the DNA?
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nothing
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When does repication of different chromosomal regions occur?
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at different times during S phase
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Hb lepore mutation
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-causes delta and beta to be fused together, so the origin cannot fit between them as it would in a normal beta-globin gene
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Identification of DNA replication origin in yeast experiment
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-two plasmid vectors containing HIS gene that is needed to make histidine
-one plasmid contains random DNA segment, oter contains ARS -plasmids introduced into yeast cells without HIS gene and when grown in media lacking histidine, first plasmid doesn't grow as well as second since ARS gene needed for good transformation -ARS is replication origin |
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What is the function of Abf1 and what is it?
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to loosen nucleosome dNA-interatction to bring in histone acetyltransferases or chromatin-remodeling complexes; it is a transcription factor
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ORC
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-origin recognition complex
-6 subunits (Orc1-6) that bind to ARS |
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DNA replication licensing system in fission yeast
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-ORC binds during early G1
-Cdt1 and Cdt6 bind to ORC during mid G1 -Helicase (MCM) bindns to either side of ORC during late G1 -Ctd1 and 6 dissociated before S phase -ensures that it is not over replicated and that it only replicates once during S phase |
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What happens to the Cdt after they are degraded in S phase?
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recycled
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What do telomeres help do?
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end replication and fill the sequence completely after RNA primers are removed
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What synthesizes telomeres?
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telomerase
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telomerase
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reverse transcriptase, and a proein complex and RNA group
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What is telomerase RNA?
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template for repeating telomere sequence as it basepairs with existing repeat
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What does telomerase use as a guide when synthesizing DNA?
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RNA
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What are the functions of telomeres?
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-protect chromsome ends from nucleases
-prevent chromosome ends from fusing together -connect chromosomes to nuclear matrix/envelope |
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Telomere synthesis
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1. lagging strand is incomple during relication, so telomerase binds and extends 3' end
2. lagging strand is completed by DNA polymerase 3. telomere sequence remains |
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telomere loops
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-larger loop is T loop and embedded loop is D loop
-embedding the loop prevents the cell from recognizing an unchecked strand and causing problems |
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Where is telomerase produced in mammals?
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eggs, sperm, and a few stem cells
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What happens to telomeres in most somatic cells?
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they get shorter with every cell division
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Telomeres in stem cells
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-always added, so never shorten
-stem cell divisions always creates one stem cell and one non-stem cell i nwhich telomeres shorten -stem cells can turn on TERT and live forever since telomeres never shorten |
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Why do telomeres shorten in somatic cells?
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TERT, telomerase reverse transcriptase, is not expressed
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What happens in tisuse culture cells when telomeres get too short?
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chromosomes fuse their ends together and cells arrest growth
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cell senescence
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-fibroblasts from a newborn divide 80 times then stop
-from 40 year old divide 40 times -from 80 year old divide 30 times -older a person is, less cells can divide |
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immortal cells
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have active telomerase
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Werner's syndrome
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-caused by inactivation of WRN gene, which encodes a helicase
-WRN helicase may be required for telomere replication -telomeres prematurely shorten, showing that telomere replication and aging are linked |
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Types of damage to DNA bases
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1. hydrolysis
2. methylation 3. oxidative damage |
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Hydrolysis of DNA bases
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-causes depurination at the rate of 5000 baes per day in the human cell
-causes deamination (removal or amine group) at rate of 100 bases/day in humans |
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Formation of thymine dimers
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-UV light can induce a change in the covalent bonds between adjacent pyrimidine bases to create a dimer between two thymine bases
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Bacterial mismatch repair
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1. MutS/MutL scan DNA for mismatches
2. When mismatch is found, MutL recruits endonuclease MutH that nicks the newly-replicated strand (no adenine methylation yet) 3. helicase removes the dNA strand and DNA polymerase fills in the single stranded DNA region |
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Eukaryote mismatch repaire
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-different MutS homologs that are specialized for single-base mismatches or for large insertions and deltions; function with different MutL homologs
-exonuclease involved in nicking is unknown |
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Nucleotide excision repair
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-RNA pol has an associated protein (CSB) that detects damaged bases
-transcription is stopped and helicases open up DNA on both sides of damage -endonucleases cleave DNA -DNA strand repaired by pol-delta and closed by DNA ligase |
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Can nucleotide excision repair occur without transcription?
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yes, but XPC is used instead of CSB
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Base excision repair
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-altered bases removed by glycosylase
-endonuclease creates nick in DNA -pol-beta binds and has a phosphodiesterase activity that removes the remaining sugar phosphate from the DNA backbone, then inserts a nucleotide -DNA ligase closes the nick |
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Pol-beta
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-has two domains: a lyase that removes damaged nucleotide and a polymerase that adds bases to DNA
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How are double strand breaks repaired?
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1. homologous recombination
2. nonhomologous end joining |
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homologous recombination
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-used after S phase when a newly synthesized sister chromosome can be used in the recombination reaction to produce a full repair
-nucleotides lost due to degradation from ends and ends are processed and homologus recombination occurs to repair the damage accurately using information from the sister chromatid |
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non-homologous end joining
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-used during G1 or G0 phase when there is no sister chromsome to pair for recombination, resulting in losee of dNA sequence
-nucleotides are lost due to degradation from the ends, and the ends join |
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How many phosphates are lost during RNA polymerization?
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2
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If RNA polymerase moves from left to right, which strand is the template strand?
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Bottom
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If RNA polymerase moves from right to left, which strand is the template strand?
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Top
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upstream
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to the left of the start site
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downstream
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to the right of the start sight and is transcribed towards 3' end
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transcription
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converts DNA template strand to RNA strand that will later be translated into protein
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What are the promoter elements?
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BRE, TATA, INR, DPE
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What are the two most important promoter elements?
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TATA and INR
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TATA location
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-30 (upstream of start site)
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DPE location
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30 downstream of start site
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INR location
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at transcription start site
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BRE location
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-35 (upstream)
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BRE transcription factor
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TFIIB
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TATA transcription factor
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TBP
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INR/DPE transcription factor
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TFIID
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How can transcriptional control elements be mapped?
|
linker scanning
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bacterial RNA polymerase subunits
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-beta
-beta prime -alpha I -alpha II -omega |
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yeast polymerase II subunits
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-RPB2
-RPB1 -3, 5, 6, 8, 9, 10, 11, 12 |
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Formation of pre-initiation complex
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1. TFIID binds to TATA box
2. TFIIA binds to promoter-TFIID complex 3. TFIIB binds to TFIIA-TFIID complex 4. TFIIF is bound to RNA Pol II 5. TFIIE binds complex 6. TFIIH binds complex |
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TFIID
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-complex of TBP and TBP-associated factors (TAFs)
-TBP is only basal transcription factor that binds specifically to DNA |
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TFIIA
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-strengthens TFIID binding to TATA box and keeps inhibitors from binding TFIID
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TFIIB
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-binds RNA Pol II, bringing it to promoter and positioning it over the start site
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TFIIF
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-helps recruitment of RNA Pol II to promoter
-destabilizes non-specific RNA Pol II-DNA interactions |
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TFIIE
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-recruits TFIIH to ocmplex
-modulates TFIIH helicase, ATPase, and kinase activities |
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TFIIH
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-helicase and Ser/Thr kinase required for initiation
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What happens when TBP binds to TATA box?
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bends DNA
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transcription initation
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formation of first phophodiester bond
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When does transcription initiation occur?
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when TFIIH (helicase)unwinds the DNA
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When does promoter clearance occur?
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when TFIIH (kinase) phosphorylates the Carboxyl-Terminal Domain (CTD) of RNA Pol II; this dirsupts the protein interaction of RNA Pol II with TBP
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When does transcription elongation occur?
|
once RNA Pol II has cleared the promoter; 5 elongation factors bind it and increase its speed 10 fold and suppress pausing and arrest
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When does transcription termination occur?
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after cleavage of RNA chain for addition of poly-A tail
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What happens after RNA Pol II comes off DNA?
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CTD-specific phosphatase dephosphoryolates it so that it can be incorporated again into a preinitiation complex
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What type of RNA does RNA Pol I transcribe?
|
pre ribosomal RNA
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What type of RNA does RNA Pol II transcribe?
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-mRNA for protein coding
-snoRNA used to process and chemically modify rRNAs -some snRNA that fucntion in nuclear processing including splicing |
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What kind of RNA does RNA Pol III transcribe?
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-tRNA
-5S rRNA gene -some snRNA |
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If RNA Pol I is mutated in yeast, what happens?
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Cell dies
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If RNA Pol I is mutated, what can be done for the cells to live?
|
given a plasmid with pre-rRNA transcribed by the Pol II promoter
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RNA Pol I initiation complex
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-upstream activating factor binds to upstream element
-core factor, TBP, and other factors bind to core elemnt -Pol I and Rrn3p added |
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RNA Pol III initiation complex
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TFIIB contains TBP, which binds with TFIIC and polymerase III to form the initiation complex
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Where are TFIIB promoters located?
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Within the coding region
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Which is more differentiated: a lymphocyte or a neuron?
|
Neuron
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What does totipotent mean and what types of cells are totipotent?
|
-a single cell can divide and produce all the differentiated cells in an organism
-somatic cells |
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What experiment shows that somatic cells are totipotent?
|
-epithelial cells from oviduct placed next to unfertilized egg cell with chromosomes removed and electric pulse sent through, donor cell will fuse with enucleated egg cell
-reconstructed zygote formed thta will create an embryo that can be placed in a foster mother |
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enhancers
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DNA sequences that are binding sites for transcription factors
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"action at a distance"
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enhancers can function thousands of nucleotide pairs away from the promoter
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What makes a DNA recognition code?
|
hydrogen bond acceptors and donors
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What type of control mechanisms do prokaryotes have, and what does this mean?
|
-negative
-genes are off until needed -regulators needed or else genes would be on |
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What type of control mechanism do eukaryotes have, and what does this mean?
|
-positive
-genes are always on -if no regulators, genes would be off -genes are on because of complex DNA folding that keeps the sequence out of reach |
|
|
helix-turn-helix motif
|
-recognition helix containing a COOH end
-turn -another helix with NH2 end |
|
|
How do helix-turn-helix motifs bind?
|
as dimers to two major grooves
|
|
|
What do helix-turn-helix homodimers recognize?
|
inverted repeats (ex: lambda cro dimer)
|
|
|
homeodomain proteins
|
specialized HTH proteins that regulate development
(ex: wild type and antennapedia mutant) |
|
|
Cys-Cys-His-His Zn Finger
|
-alpha helix and beta sheet held together by zinc
-binds as a monomer because each motif can contact the DNA (high bonding affinity) |
|
|
C4 zinc finger
|
-two alpha helices held together by zinc
-only one of the alpha helices fits into the major groove, so bind as dimers usually |
|
|
Where are C4 zinc fingers found?
|
in steroid hormone receptors which have only 2 zinc fingers per protein
|
|
|
leucine zipper
|
-amphipathic alpha helix with leucines spaced every seven amino acids in the dimerization motif
-entire protein is alpha helix that forms dimer through leucine zipper domain -protein binds DNA with each alpha helix lying across the major groove |
|
|
helix-loop-helix
|
-alpha helix and short loop of amino acids, then another helix
-binds DNA as a dimer -one alpha helix lies across major groove and other forms dimer similar to leucine zipper |
|
|
What is a positive aspect of heterodimerization?
|
can expand DNA binding repertoires
|
|
|
What do dominant negative dimer partners do?
|
inhibit activity
|
|
|
DNA affinity chromatography to isolate sequence-specific DNA binding proteins
|
-total cell proteins put in column with matrix containing DNA from many differnt sequences
-low salt wash removes proteins that do not bind to DNA -medium salt wash elutes many different DNA binding proteins -if put in column with matrix containing only GGGCCC, medium salt-wash removes all proteins not specific for GGGCCC and a high salt wash elutes rare proteins that specifically recognize this sequence |
|
|
Identification of binding consensus sequence of a transcription factor
|
-gene protein of unknown DNA-binding specificity is introduced to a large pool of shot DNA double helices that have randomly generated nucleotide sequences
-protein-dNA complexes sepaarted from free DNA using gel mobilit short assay -remove protein and determine sequences of tightly bound dNA fragments -this produces DNA consensus sequence |
|
|
What is used to identify genomic regions boud by transcription factors?
|
Chromatin imunoprecipitation (ChIP)
|
|
|
How does ChIP work?
|
-living cell contains 2 genes with 2 proteins
-proteins are cross-linked to DNA with formaledhyde, the cells are lysed, and the DNA is broken into small fragments -DNA precipitated using antiboides against the first protein then the reverse formaldehyde cross-links removes the protein -precipitated DNA is amplified by PCR then the DNA corresponding to those positions in the genome that were occupied by the ptorein in the cells can be isolated |
|
|
How do transcription factors work?
|
-bind to binding to domains to turn genes on
-must be correct binding domain (ex: LexA Gal4 cannot bind to Gal4 domain) |
|
|
How do transcription factors interat with the basal transcription complex?
|
through DNA loops
|
|
|
When does a DNA loop form?
|
-when the polymerase is in a closed complex on promoter and ATP has been used, enhancer creates looped activiation intermediate that allows the polymerase to start working and turn the gene on
|
|
|
What does a mediator do?
|
helps assemble and stabilize transcription factors/etc, load RNA Pol II, brings in proteins
|
|
|
Yeast mediator-Pol II complex
|
-mediator complex binds RNA Pol II and facilitates its loading onto the promoter
-contains over 20 proteins, including a HAT -associates with chromatin remodeling proteins |
|
|
How can transcription factors facilitate the switch from heterochromatin to euchromatin?
|
-condensed (heterochromin) when activated gets decondensed (euchromatin) which turns the gene on due to a mediator-pol II complex
|
|
|
How does acetylation of histones facilitate transcription?
|
-mediator contains HAT activity
-acetylation activates chromatin due to interaction between activator and coactivator -facilitates TFIID binding to DNA -HAT binds to nucleosomes and a second activator protein binds which enables the TFIID to bind to DNA |
|
|
Modes of action of transcriptional repressors
|
1. competitive DNA binding
2. masking the activation surface 3. direct interaction with the general transcription factors 4. recruitment of repressive chromatin remodeling complexes 5. recruitment of histone deacetylases |
|
|
Competitive DNA binding for repressing
|
repressor binds to binding site for repressor, which is overlapping binding site for activator, so the activator cannot bind
|
|
|
masking the activation surface for repressing
|
both repressor and activator bind at binding sites, but also bind together, so activator can't work
|
|
|
direct interaction with the general transcription factors for repressing
|
both activator and repressor bind, but repressor forms a loop and binds to TFIID, so activator cannot bind
|
|
|
Positive feedback loops
|
-when cells undergo a cell fate decision to become a particular type of cell, it usually involves expression of master transcripitonal regulators
-they turn-on their own expression so they are expressed in all subsequent daughter cells and turn-on expression of genes required for that cell fate and/or turn off expression of genes not needed for that cell fate |
|
|
What are some ways transcription factors are activated?
|
-protein synthesis
-ligand bonding -protein phosphorylation -addition of DNA binding subunit -unmasking by phosphorylating inhibitor -stimulation of nuclear entry -release from membrane |
|
|
What does methylation of a cytosine in a CG sequence do?
|
inhibits gene expression and transcription
|
|
|
Methylation of DNA
|
recruits histone deacetylases and chromatin remodeling complexes that repress transcription
|
|
|
imprinting
|
-occurs when DNA methylation turns off genes in one of the sexes
-imprinted allele and expressed allele -both parents express same allele and during meiosis imprinting is removed -males have methylation -offspring differ in the allele that is expressed |
|
|
insulators
|
-limit the influence of enhancers on promoters
-enhancer can't work past an insulator to express a gene |
|
|
What are some modifications of hnRNA to make it mRNA?
|
1. capping of 5' end
2. addition of poly-A tail 3. splicing to remove introns |
|
|
hnRNA
|
heterogenous RNA
|
|
|
When is a 5' cap added to RNA?
|
after the elongation factor has bound to RNA Pol II
|
|
|
When does transcription elongation stop?
|
When it reaches the poly-A addition sites
|
|
|
What happens to uncapped mRNA?
|
it is rapidly degraded and termination occurs
|
|
|
What happens to capped mRNA after it is cleaved off?
|
poly-A tail replaces the elongation factor
|
|
|
Where do RNA modifying enzymes reside?
|
on RNA Pol II CTD
|
|
|
What must first happen to a strand before it can be capped?
|
it must be phosphorylated
|
|
|
Are all RNAs 5' capped?
|
No, only those transcribed by RNA Pol II (not I or III)
|
|
|
Where is the 5' cap added on the RNA strand?
|
Within 30 nucleotides of the start of transcription
|
|
|
What exactly is the 5' cap?
|
a 7-methylguanosine added to a 5'to5' triphosphate bridge located at the 5' end of the primary transcript
|
|
|
How does poly-A addition occur?
|
-you have an AAUAAA start, followed by a 10-30 nucleotides and a CA; then less than 30 nucleotides and a Gu or U rich region
-cleavage betwen CA and less than 30 nucleotide region -OH group onto CA -GU or U rich region degraded in nucleus -poly-A addition occurs after the CA but before the OH |
|
|
What side is the poly-A tail added to?
|
3' end
|
|
|
How many adenosines are in the poly-A tail?
|
200-250
|
|
|
Do all RNAs have poly-A tails?
|
No, only if transcribed by RNA Pol II
|
|
|
Do all mRNAs have a poly-A tail?
|
Yes, except histone mRNA, which has a stem-loop structure at the 3' end
|
|
|
If a gene normally transcribed by Pol II is placed behind an RNA Pol I or III, will it recieve a cap or tail?
|
No
|
|
|
hnRNP
|
heterogenous ribonucleoprotein particles; form when hnRNA transcripts are coated with proteins
|
|
|
What does an hnRNP particle consist of?
|
about 500 nucleotides of RNA wrapped around a protein core
|
|
|
What is the function of hnRNP thought to be?
|
to package RNA to keep it from forming secondary structures
|
|
|
Where did the evidence from introns come from?
|
from hybridization studies of genomic DNA and mRNA analyzed by electron microscope
|
|
|
Where does RNA splicing occur?
|
in the nucleus
|
|
|
When does splicing occur?
|
While RNA is still being transcribed
|
|
|
The ___ splice site is recognized and paired with the next synthesized ____ splice site
|
5'; 3'
|
|
|
When can mRNAs be sent to the cytoplasm for translation?
|
only once they are fully spliced
|
|
|
Introns have a few ___________ near their ends
|
conserved sequences
|
|
|
What size are introns?
|
can range from 50 ntds to 10000 ntds
|
|
|
__________ site is very important to splicing
|
branch point A
|
|
|
Introns are flanked by...
|
5' exon sequence and 3' exon sequence
|
|
|
An intron sequence includes a _______ and a ______
|
branch point A and 2'OH
|
|
|
lariat structure
|
formed by looping of the intron and causes the 5' intron side to bind to the A site and the OH transfers to the end of the 5' exon
-one side of the intron is still attached to the 3' exon so the OH transfers to the lariat structure and the two exons splice together |
|
|
What is involved in splicing?
|
-5 U-rich small nuclear RNAs (snRNAs): U1, U2, U4, U5, U6, each packaged with 6-10 proteins to form small nuclear ribonucleoproteins (snRNPs) that form a spliceosome
|
|
|
How many ATPs are used during the splicing of one intron?
|
3; used to facilitate snRNP rearrangements
|
|
|
How does RNA splicing process work?
|
-initial pre-mRNA transcript has a BBP and U2AP on the A site
-U1 and U2 snRNP replace the BBP and U2AP -U1 binds to the beginning of the intron while U2 binds to the A site -u4/U6 and U5 snRNP attaches to bind and draw the two ends together to form a lariat and a 5' splice site cleavage -U1 and U4 are removed -U6 snRNP is still attached to 5' exon and it binds to the 3' exon end, releasing the intron and joining the exons -intron degraded and snRNPs recycled |
|
|
What are the two major reasons for introns?
|
-exon shuffling can create new gene combinations
-alternative splicing in which one gene can produce different proteins (increases number of different proteins each gene can encode) |
|
|
consitutive alternative splicing
|
certain splice sites are inherently ambiguous to the spliceosome, so sometimes it is chosen and other times it is not
|
|
|
regulated alternative splicing
|
certain splice sites are used at certain times or in certain cell types (positive or negative)
|
|
|
negative regulation (splicing)
|
protein factor binds and masks a splice site so that another site is chosen
|
|
|
positive regulation (splicing)
|
protein factor directs spliceosome to an overlooked splice site
|
|
|
How is alpha-tropomyosin spliced?
|
spliced differently in each type of mRNA (smooth muscle, fibroblast, striated muscle)
|
|
|
Who discovered self-splicing?
|
Tom Cech in tetrahymena
|
|
|
Tom Cech's experiment
|
-isolated rRNA from tetrahymena and found that it spliced without adding proteins
-transcribed rRNA from DNA with bacterial polymerase and found that it had never been associated with any eukaryotic enzymes and also self-spliced |
|
|
Type I self-splicing introns
|
-uses a free G nucleotide to attack 5' splice site initially
-breaks bond -3' splice site is cleaved and the exons ligate |
|
|
Type II self-splicing introns
|
-uses internal A residue to attack 5' splice site
-3' splice site cleaved and forms lariate structure -exons ligate |
|
|
What has type I self-splicing?
|
some nuclear genes of protozoa, plant chloroplast genes, mitochondrial genes of fungi
|
|
|
What has type II self-splicing?
|
some mitochondrial genes of fungi and plant chloroplast genes
|
|
|
Does self-splicing occur in vivo?
|
Type I does, but type II only in vitro at elevated temperatures and high Mg concentration
|
|
|
RNA World model based on catalytic RNAs
|
-early cells used RNA as genetic material and RNA instead of proteins for catalysis
-initially, only RNA and it produced protein, which in turn produced DNA, then more RNA |
|
|
What is involved in active transport into the nucleus?
|
-all nuclear proteins (DNA & RNA pols, histones, transcription factors, snRNPs)
-ribosomal proteins |
|
|
What is involved in active transport out of the nucleus?
|
-mRNA
-tRNA -snRNA -ribosome protein/RNA subunits |
|
|
What are the two types of nuclear localization signals?
|
-simple NLS
-bipartite NLS |
|
|
Simple nuclear localization signals
|
-high percentage of positively charged amino acids within a stretch of 7-10aas
-required -can get proteins into the nucleus |
|
|
Bipartite nuclear localization signals
|
-2-10 positive amino acids
-3 of 5 positions |
|
|
What happens if you add a nuclear localization signal to a protein?
|
can direct them to the nucleus
|
|
|
Nuclear pore have _______ symmetry
|
octagonal
|
|
|
Structure of nuclear pore
|
contains nuclear basked, spokes, cytoplasmic filaments
|
|
|
Mechanism of nuclear import
|
-Importin (alpha and beta) bind NLS on cargo and transport it to the nucleus
-in the nucleus, Ran-GTP binds importin, causing the release of cargo -importin-Ran-GTP is transported to the cytoplasm -in cytoplasm, Ran-GTP converted to Ran-GDP by GTPase activating protein Ran-GAP -Ran-GDP transported to nucelus where GEF converts it to RAN-GTP |
|
|
Mechanism of nuclear export
|
-exportin + Ran-GTP bind cargo with a nuclear export signal in the nucleus
-complex transported to cytoplasm -in cytoplasm Ran-GTP converted to Ran-GDP by Ran-GAP and complex dissociates -exportin and Ran-GDP are transported to the nucleus -in the nucleus GEF converts Ran-GDP to Ran-GTP |
|
|
What do chromosomes require?
|
1. origins of replication
2. centromeres 3. telomeres |
|
|
What is the major function of chromosomes?
|
To provide genes
|
|
|
structural RNAs
|
-tRNA
-rRNA |
|
|
bioactive RNA
|
catalytic RNA (including rRNAs)
|
|
|
In higher eukaryotes, do most of the DNA encode for genes?
|
No
|
|
|
heterochromatin
|
inaccessible to transcriptional machinery
|
|
|
euchromatin
|
accessible to transcriptional machinery which includes actively transcribed genes
|
|
|
Where is rRNA synthesized?
|
nucleolus
|
|
|
How are chromosomes kept in the proper place?
|
by Matrix Association Regions (MARs) on DNA that bind to the nuclear matrix
|
|
|
What are some types of genes?
|
-solitary genes
-duplicated and diverged gene families -tandemly repeated genes |
|
|
What are some repetitious DNA?
|
-satellite DNA
-interspersed repeat DNA |
|
|
unclassified spacer DNA
|
arises from repetitious DNA that break down over time and become indistinguishable
|
|
|
What does gene duplication allow?
|
-functional divergence
-if no gene duplication and a mutation occurs to change the function, it dies -if duplicated, more to not mutate and stay alive |
|
|
Evolution of the globin gene family
|
-duplication of ancestral gene allowed the specialization of different alpha and beta-globins for different life stages: fetus, embryo, and adult, when requireements of hemoglobin for obtaining oxygen are different
-fetal globins have higher affinity for oxygen so they can collect from mother in placenta |
|
|
Gene duplication is thought to arise from:
|
1. inappropriate joining of chromosome breaks
2. unequal cross-over catalyzed by repetitive DNA sequences 3. transposons flanking gene that transport the gene to a new site |
|
|
Renaturation
|
-used to measure repetitive dNA
-chromosome is broken into 1000 base pair fragments and they are denatured -then allowed to hybridize and the rate of renaturation is measured to determine the amount of repetitive DNA -first to associate is highly repeated, longer to renature, less amount there |
|
|
Tandemly repeated genes
|
-found next to each other in the same orientation
-gene duplicates in the same chromosomal location and can turn into tandem repeats by unequal corssin-over between sister chromosomes -identical sequences can be kept in each repeated gene by unequal crossovers and gene conversion |
|
|
satellite DNA
|
-simple repeat DNA
-constitutes 10-15% of genomic DNA -generally 5-10 base pair tandem repeats -generally associated with centromere regions |
|
|
How many satellite DNA repeat species in humans?
|
more than 10
|
|
|
Does satellite DNA have a function?
|
has not been shown
|
|
|
Satellite DNA evolves ______
|
rapidly
|
|
|
Why is it called satellite DNA?
|
forms satellite bands to main DNA due to base composition when put in CsCl equilibrium centrifugation
|
|
|
minisatellite DNA
|
found in 1 to 5 kb regions containing repeats of 15 to 100 bps
|
|
|
How is minisatellite DNA used in DNA fingerprinting?
|
differences in length
|
|
|
microsatellite DNA
|
-found in 50-100 bp regions with repeats of 1-5 bps
|
|
|
Trinucleotide microsatellite DNA found to be associated with what?
|
Huntington disease, mytonic dystrophy, fragile-X mental retardation; genetic anticipation-symptoms get more sever due to lengthening of repeats
|
|
|
DNA fingerprinting
|
-DNA obtained
-enzyme digests DNA and results run on gel with probel with radioactive repeat DNA -DNA that shows up on the band are different lengths of minisatellites -identical minisatellite DNA sequenes throughout the genome, so you get multiple bands |
