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Why does it matter that DNA would be in (right-handed) B-form than in A form?
It is called B form due to the twists in the coil being of a particular distance, longer than the A-form. This has implications for the recognition of which proteins may have been acting as transcription factors (their size has to be such that they’d fit into the groove and therefore properly react with the DNA). In fact, the researchers who first crystalized dsDNA had dehydrated it a little too much and thus compressed the twists, reporting dsDNA was in A form at first. As a result of this error, certain proteins were initially disregarded as potential transcription factors when they in fact were such.
dsDNA can change to ssDNA simply by heating it up. Why?
The temperature dependent of GC content because AT hydrogen bond to eachother with 2 bonds, whereas GC are with 3 bonds. The more GCs, the stronger the strands of DNA.
What are the important regions of chromosomes?
Origins of replications, Centromeres, Telomeres
Replications of origins (Ories):
regions where DNA polymerases and other proteins bind to initiate DNA synthesis (scattered throughout a eukaryotic chromosomes since they are so long. But bacteria had typically one or two ories; they are shorter and circular)
condensed central regions to assist in segregation during cytokinesis. Kinetocores form here too
- required for the stability of chromosomes and for starting the process of chromatin condensation
- help prevent the length of chromosomes Via Repeats of simple sequences. Something to do with lagging strands being short and not affecting the region of DNA coding for the gene., doesn’t get cut off. Keeps adding nucleotides so that it doesn’t get shortened.
- Also allows for the Chromatin Remodeling proteins to bind, condense Chromosomes, like HDAC
- This means that telomeres also regulate/suppress transcription
regions where the DNA is moderately or extensively condensed. The more condensed, the less it will be transcribed. Only in eukaryotes. Preferentially located at the periphery of the nucleus, and we don’t know why.
- poor expression of genes—silenced, nearly no transcription.
- in regions of telomeres and Y chromosome
moderately transcriptionally active region of chromatin can more easily transition to euchromatin which is very transcriptionally active
For DNA to decondense:
first the nucleosomes disassociate from one another and then the histones disassociate from the DNA. DNA then opens up and forms euchromatin
For histone acetylation:
Needs histone acetyl transferase enzyme (HAT). Decondenses DNA so it can be pulled through RNA polymerase
For histone deacetylation:
HDAC, histone deacetylase enzyme. Condenses DNA
RAP 1 :
a protein that has DNA binding domain, only binding to telomeres and has a protein interaction domain, initiating process of silencing transcription
Deacetylation of Histones:
Includes RAP1. Then SIR 3 and 4 (Silent information regulator 3 and 4), Have protein interaction domains which bind to eachother and RAP 1. Then the same interaction domain binds to SIR 2 (an HDAC). Then deacetylation of tails occurs. Heightens positive charge. Then more SIR3/4 binds to histone tail and it continues on down to the next histone/nucleosome.
The histone code:
A predictable pattern that drives the expression of genes; combination of possible combinations of modifications
Transposable elements (TE): 2 types moderately repeated element
- Retrotransposon: (RNA transposable elements) two step process (copy-and-paste) requires RNA intermediate. RNA polymerase makes an RNA transcript from a DNA sequence which is then reverse transcribed in the nucleus to make a DNA intermediate which is then inserted in another site in the genome. Requires reverse transcriptase.
- DNA transposon (jumping gene, multi-colored corn kernels)one step cut and paste process
IN what phase does DNA replication occur:
How do we know that mutations are repaired?
Because DNA polymerase makes a mistake about 1 nucleotide per 10^4 polymerized nucleotides, but the fixed error rate is 1 in 10^9, meaning that something is fixing
Proofreading by DNA polymerase that occurs during DNA replication:
The loading of an incorrect nucleotide is sensed by the DNA polymerase while it tries to move forward due to the incorrect hydrogen bonding. Thus an exonuclease activity of the DNA polymerase will excise the incorrect nucleotide
Mut S scans for the bulge in the DNA arising from the mismatching of the nucleotides. It then recruits Mut L, which scans for a nick in the DNA (lack of a phosphodiester bond) in the newly synthesized strand of DNA; it then degrades the nicked strand back to the mismatch and allows for the new gap to be filled in by DNA polymerase
nonhomologous end joining:
enzyme comes in to cleave off the ends and DNA ligase brings the strand together again.
- After the cleaving off of the ends at the double strand break, the missing sequence is brought in from the sister chromatid and is processed to repair the damage, without the deletion of any genetic sequence
- Broken DNA is cleaved up by exonucleases to provide broader overhangs, providing a free 3’ hydroxy that the DNA polymerase can polymerize off of. The protein RAD 51 then drags the broken strand into a homologous chromosome until it finds the complementary sequence. DNA polymerase then uses the homologous strand as a template to copy and it continues along the unbroken strand. The ends are then ligated via DNA ligase and produces two strands of DNA which are a mixture of each sister chromatid
- TATA box binding protein (TBP) connected to Transcription factor II D (TFIID, which will bind and bends DNA).
- TBP binds to TATA.
- TFIIB Adds.
- Remaining TF and RNA polymerase assembles at promoter
- TFIIH (helicase) unwinds DNA
- Initially, RNA polymerase to stays at the promoter to make short strands of RNA. Then TF2H acts as a kinase, and phosphorylates RNA polymerase two, So it can begin transcription.
- The phosphorylation of RNA polymerase two occurs at the Carboxy Terminal Domain of RNA polymerase two (for Eukaryotes). CTD is made up of many (26+) repeats of the Hepta peptide. As if he RNA polymerase two Transcribes, CTD becomes more phosphorylated, increasing transcription rate to a limit. This causes the disassociation of initiation complex proteins and the Association of mRNA processing proteins, such as capping enzymes and spliceosomes. Once it reaches the end of the gene it disassociate from the DNA and phosphatases come in to dephosphorylate the polymerase so that it can reinitiate.
RIBOSOME ARE MADE OF Protein and rRNA
- They have four binding sites, 3 are for tRNAs at the A, P and E-sites.
- A: aminoacyl-tRNA; Site where an amino acid bound to the tRNA is added to the ribosome
- P:peptidyl-tRNA; Site where the amino acid is transferred from the tRNA to the peptide (polymerization site)
- E: Exit; Where the tRNA is ejected from the ribosome
splicing, polyadenylation, and capping
Take DNA, expose it to DNase, and let all of the DNA that is not bound to this transcription factor degrade Because DNase cannot get access to DNA that is bound to another protein. Now you can isolate and sequence the DNA (sequence the peptide as well)
Gel shift assay:
To tell if DNA will bind with a transcription factor, take one sample that is bound to the transcription factor and one sample that is not and run a gel. If it does interact with the transcription factor, it will travel less.
What are four ways a genome can change?
Intragenic mutation; Gene duplication; DNA segment shuffling; horizontal transfer Between two organisms
What type of bonds and bond nucleotides together?
From what to what is the primary proteins structure usually written from left to right?
From the N terminus or any no terminus to the C terminus, or carboxyl terminus
What bonds provide structure to proteins?
The disulfide bonds between Cystine amino acids
What is the Proteosome?
A multi unit protein complex which unfolds Polly – ubiquitinated proteins and cleaves them into peptides, Which are then furthered degraded by cytosolic Peptidases into amino acids
What nucleotides does DNA have? RNA?
DNA: adenine, guanine, cytosine and thymine. RNA: adenine, Guanine cytosine and uracil
What determines the temperature at which double-stranded DNA will melt into single-stranded DNA?
The GC content
What are the three functional elements of chromosomes and what do they do?
- Replication origins: regions where DNA polymerases and other proteins bind to initiate DNA synthesis… Scattered throughout the eukaryotic chromosome
- Centromere: constricted region required for proper segregation via the kinetochore
- Telomeres: located at both ends of each chromosome for stability and YAC
genetic material made up of DNA and associated proteins
Octomers of histones around which DNA wraps
What are three ways in which that Histone tails can be modified to affect how tightly they bind to DNA?
Acetylation, methylation, and phosphorylation
What does that histone code entail?
The combination of possible keystone modifications. It regulates histone function in a predictable manner, changing the binding sites for DNA associated proteins
HAT does what?
Histone acetyl transferase enzyme; neutralizes positive charge on histone tails, therefore decreases histone unbinding, therefore transcription begins
HDAC does what?
Remove acetyl groups from tails, causing stronger binding to DNA, condensing DNA
What are the methods of DNA repair?
DNA polymerase proofreading activity; TG mismatch repair; mismatch excision repair; thymine dimer repair; breaks in DNA by: enjoining or homologous recombination
mediated by exonuclease activity of DNA polymerase (Some RNA polymerases can proofread too)
- Mut S finds the mismatch based pair
- MutL scans for Nick; then degrades
- me back to mismatch.
- Gap is filled by DNA polymerase.
- Same strategy for TT dimers (Joined by UV light)
- Exonuclease provides three prime oh. Rad 51 invades homologous DNA.
- Three prime end extended by DNA polymerase and base pairs with unbroken
- strand. Three prime end of broken strand extended using invading strand
- as Template. Repaired ends ligated. Strands are Cleaved
- Activator Sw15 binds enhancer sites (1000+Bp Upstream of start site)
- Then interacts with chromatin remodeling complex (SwI/Snf) to decondense chromatin, exposé histone tails
- Complex containing histone acetylase associates and adds acetyl groups (further decondense)
- New associated complex helps SBF activator bind to DNA near promoter
- SBf recruits mediator
- RNA polymerase 2 and general transcription factors bind
Control regions more 200 base pairs from start site
- control regions 100 to 200 base pairs from start Site
- Cell type specific
- 25 to 35 base pairs upstream of start site
- Positions RNA Polymerase two for transcription
Heterogenous ribonucleoprotein; RNA version of TF, with RNA binding and protein interaction domains
- 1. Capping enzyme (Phosphatase) removes terminal P from 5’ end
- 2. CE (Guanylyl transferase): Adds GTP to remaining 5’-diphosphate, removes PP
- 3. Guanine-7-methyl transferase methylates G at end (N)
- 4. 2’O-methyl transferase methylates G in middle (O)
Describe the roles of the all of the RNAs in the fully formed spliceosome complex that is interacting with mRNA.
- The RNAs making up the spliceosome complex are termed small nuclear RNAs (snRNAs); loaded with uracil residues, they are known as U1, U2, U4, U5, and U6. U1 identifies and binds to the sequence of the 5'-splice site of an intron in a strand of pre-mRNA while U2 complementarily binds to the 3' splice site. This binding exposes an unpaired adenosine such that a nucleophilic attack may occur at the intronic 5' splice site, eventually incurring a removal of the intron and a joining of the exons in the mRNA altogether. (U2 may also function to join two separate RNA molecules in a like role in trans-splicing when it binds the exon of one RNA to another with the help of an SL1 snRNP, which removes a splice site and leads to the exposure of an 'outron.') U4 works to regulate U6 within their complex; when the spliceosome rearranges, U4 (as well as U1) is released, by its nature of ATP-dependence manner causing realignment of the U6 to expose the active site for splicing catalysis. Therefore U6 and U2 are known as the catalytic core for the transesterification that results in the ligation of the two exons/release of the intron.
- U5 remains with U4/U6 from begining, ends with U6 and U2.
- CPSF to Poly A
- CStF, CFI CFII
- PAP (poly A polymerase)
- Cleavage by CFI and CFII
- PAP Adds As (positive feedback)
- Nucleotide substitution
5'cap, Polyadenylation, Spilcing, Editing
- heterodimer of TAP (nuclear
- export factor 1) and Nxt1
- (nuclear export transporter 1). TAP/NxtI interact with nucleoporins
- to help export mRNPs through the
- NPC and in to the cytosol.
Immediately after leaving the nucleus:
Nonsense-mediated decay test to test for correct mRNA splicing.
aminoacyl-tRNA synthases have three active sites:
bind aspecific amino acid, a specific tRNA, and ATP
removes amino acids that are attached to the wrong tRNA.
Ribosome: 4 RNA binding sites:
mRNA; A: aminoacyl-tRNA: where amino-acid bound to the tRNA is added; P: peptidyl-tRNA: where the amino acid is transferred from the tRNA to the peptide (polymerization site); E: exit; where the tRNA is ejected
When ribosome reaches a stop codon (UAA, UGA, or UAG), not recognized by tRNAs. eRFI and eRF3-GTP enter near A-site when ribosome nears stop codon. Then GTP hydrolysis transfers OH to peptide at P site and all is released.
Incorrectly folded proteins:
refolded by chaperones or degraded by proteasome