Important Vocab First M2M exam Flashcards
Mitogens
Mitogens and other growth factors are proteins that lead to production of Cyclin D1-3.
Enough Cyclin D1-3 will eventually activate CDK4 and CDK6 –> these will phosphorylate Rb protein.
Rb protein
Is an inhibitor of cell cycle–> particularly Synthesis of DNA.
Will be phosphorylated by CDK, which will end Rb’s inhibition of transcription factor E2F.
E2F will lead into DNA replication.
CDKN family (The Cyclin Dependent Kinase inhibitors)
o The CDKN 1 and CDKN 2 families are a group of proteins that inhibit the CDKs in cell cycle binds to CDK and competes with cyclin for the binding spots of CDK
-CDKN1 stops CDK 1-6
-CDKN2 Stops CDK 4,6
o These CDKN families are CRITICAL for normal healthy cell cycle
-P16 and p21 are very important CDKN proteins that inhibit CDK, their defect is very likely to cause cancer
-P21 works with tumor suppressor and guardian angel p53
Pre-RC formation (Pre-replication complex)
oThe replication center consists of the Orc protein
oOrc Protein 1-6= recognizes the origin site on DNA
-The Orc protein will call upon other proteins to get replication going Cdt1 and Cdt6
o Cdt1 and Cdt6 recognize Orc protein and summon Helicase
-DNA helicase will heat/cut the DNA and allow it to unwind
Regulating the Replication Complex
pre RC
o CDK protein inhibits building of the Replication complex, however it Activates it! (activates helicase specifically)
-Means that during G1 phase, CDK is low and the pre-RC is able to build
-During Synthesis CDK has ramped up and is high, stops building of pre-RC but allows it to activate!
o During Mitosis
-CDK is still high, not allowing pre-RC to build
-The replication center has already aided in DNA synthesis
Pre-Replication Complex activation
o In G1, the Orc will use Cdt1 and Cdt6 to summon helicase and have it loaded and ready
o By synthesis phase The Replication Center (helicase only) is activated by the CDK via phosphorylation pathways!
CRITICAL CDK activates the helicase that has been loaded by the pre-RC complex
o However, CDK deactivates the Orc protein and Cdt1&6
o It is critical that Orc1-6 and Cdt proteins are deactivated by CDK
Otherwise there could be more than one replication
DNA damage checkpoint proteins
o Rad17 will bind to DNA damage from radiation or UV light
-Will signal to ATM or ATR protein
o ATM/ATR protein kinases that will Inhibit CDK!
-ATM inhibits for mostly double strand DNA damage
-ATR does almost all types of DNA damage
ATM and ATR will signal to the p53 gene and the p21 gene
-These will prevent G1 Synthesis and G2 mitosis
-Does this by inhibiting the CDK
o Chk2 and Chk1 = ATM and ATR lead to both of these cascades
-ATR focuses on Chk1
Aminoacyl tRNA synthase
o Proves the amino acids for specific tRNA
Scans each tRNA to make sure it is binding to specific one using “recognition elements
This matching is crucial, since it ensures that only the particular amino acid matching the anticodon of the tRNA, and in turn matching the codon of the mRNA, is used in protein synthesis.
Due to the degeneracy of the genetic code, multiple tRNAs will have the same amino acid but different codons.
Initiation in Prokaryotes (first step of Translation)
o Prokaryotes: Rely on Shine-Delgarno sequence of mRNA for the ribosome to recognize
- Shine Delgaro: A bunch of adenine and guanines (sometimes an uracil) that the ribosome can recognize
- Critical for Shine Delgaro sequence to begin initiation, along with initiation factors
oProkaryotes continued Have three different Initiation Factors (IF1, IF2, & IF3)
- IF1 and IF3 bind to the 30s subunit, as does the mRNA due to the Shine-Dalgaro site.
- IF1 and IF3 help guide mRNA start codon AUG to the subunit’s P-site
- IF2 delivers initiator formylmethionine to the P-site of the 30s subunit
•GTP hydrolysis of IF2 releases all the initiation factors, and leads ultimately to 50s coming down to bind on the 30s, forming the overall 70s subunit
-IF2 using a GTP= the entire subunit 70s forming, and moving the codon to the A-site to be read
Initiation in Eukaryotes (first step in translation)
o Depends of the 5’ Cap (7-methyl guanine cap), and it depends on roughly 12-20 different initiation factors
- Get the 5’ Cap and the mRNA ultimately to the P site of the ribosome (codon will move to A-site after GTP use)
-Remember, the 5’ cap is exclusive to Eukaryotes
o The variety of initiation factors and overall complexity= allows for more regulation and control!
-Initation factor eIF4E is Critical for binding the 5’ of mRNA–> leadings to binding of many of eIFs and the small ribosomal subunit.
eIf-2Alpha =A critical initiation factor for translation
o Aids in the binding tRNA to the ribosome via GTP hydrolysis
Elongation in bacteria and eukaryotes
o PTC Peptidyl Transferase Center
- Is on the large subunit of the ribosome (23/28s unit)
- Is considered a ribozyme
o Energy for the peptide bond formation comes from the tRNA charging
- Peptide bond formation: leads to transfer of nascent (new) amino acid from the P-site tRNA to the A-site tRNA
- peptides are being added and creating growing chain on the end of the P-site
- Through the use of Elongation Factor 2 (EF2) and a GTP hydrolysis, the tRNA is moved from the A site to the E-site
o After EF2 uses a GTP, the empty tRNA moves to the E-site, able to exit
o A new tRNA with a peptide can now move from the A-Site to the P-site
-Each new tRNA with an AA comes with an Elongation Factor 1
Chain Termination (Third Step of translation)
o Release factors binding to the stop codon
- Stop Codon CANNOT release without the release factor proteins
mTOR=mammalian target of rapamycin
eukaryotes
o A kinase that is a Critical regulator of translation (in eukaryotes)
o Deals with many of the Initiation factors/proteins
o 4E-BP1 will aid with translation when phosphorylated= attaches to the 5’ cap
-mTOR activated and phosphorylates 4E-BP1 this than frees to attach activate the mRNA cap for translation
Kozak Sequence
o There are multiple AUG start codons in an mRNA sequence, this can lead to a variety of proteins being made, with one being more dominantly made based on its AUG initiation strength
- Kozak Sequence= GCCRAUGG , strength of this sequence will decide which protein is predominantly made on an mRNA strand
- stronger kozak sequence= more of that specific part of mRNA being translated
Cap-Independent Translation (eukaryotes)
IRES- Internal Ribosome entry sites
IRES driven Translation:
Translation without 5’ cap, needed during virus-infested translation
Allows mRNA to make necessary during tough times in the body (virus invasion)
o Unfortunately, Virus can also undergo cap independent translation (more so than eukaryotes)
-In some cases, the virus produces a protease that cleaves eIF4G, shutting down cap-dependent translation. The virus can continue using an IRES
eIf-2Alpha
eIf-2Alpha =A critical initiation factor for translation
-Aids in the binding tRNA to the ribosome via GTP hydrolysis
Eukaryotic Initiation Factor 2 (eIF2) is a eukaryotic initiation factor. It is required in the initiation of translation.
eIF2 mediates the binding of tRNAmet to the ribosome in a GTP-dependent manner. eIF2 is a heterotrimer consisting of an alpha (also called subunit 1), a beta (subunit 2), and a gamma (subunit 3) subunit.
Once the initiation is completed, eIF2 is released from the ribosome bound to GDP as an inactive binary complex. To participate in another round of translation initiation, this GDP must be exchanged for GTP.
eIF4E
Initation factor eIF4E is Critical for binding the 5’ of mRNA–> leadings to binding of many of eIFs and the small ribosomal subunit.
eIF4E is a eukaryotic translation initiation factor involved in directing ribosomes to the cap structure of mRNAs. It is a 24-kD polypeptide that exists as both a free form and as part of the eIF4F pre-initiation complex.[3] Almost all cellular proteins require eIF4E in order to be translated into protein. The eIF4E polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis.
Three prime untranslated region (3’-UTR)
Several regions of the mRNA molecule are not translated into protein including the 5’ cap, 5’ untranslated region, 3’ untranslated region, and the poly(A) tail. The 3’-UTR often contains regulatory regions that post-transcriptionally influence gene expression.
Short Read Sequencers
Next gen sequencing
DNA sequencers that produce millions to billions of short 100 base-pair reads in a single run. Machines use a 1000 base pair DNA template input.
Can read one or both sides of the DNA molecule.
These machines have much lower error rate than long-read sequencers (1 in a million roughly). Used typically for high coverage/ read depth on specific subsets of DNA.
Long Read Sequencers
Next gen sequencing
DNA sequencers that produce 10,000 sequencing reads than can reach 10,000 base pairs length. Have a much higher error rate than short sequencer. These machines focus on a single molecule approach, such as watching a single DNA Polymerase.
Read Depth “Coverage”
Higher coverage= improves confidence that true variant was identified and is not due to errors in the sequencing reaction.
Coverage (read depth or depth) is the average number of reads representing a given nucleotide in the reconstructed sequence. This parameter also enables one to estimate other quantities, such as the percentage of the genome covered by reads (sometimes also called coverage). A high coverage in shotgun sequencing is desired because it can overcome errors in base calling and assembly. The subject of DNA sequencing theory addresses the relationships of such quantities.
Sometimes a distinction is made between sequence coverage and physical coverage. Sequence coverage is the average number of times a base is read (as described above). Physical coverage is the average number of times a base is read or spanned by mate paired reads
Exome Sequencing
Exome sequencing is a technique for sequencing all the protein-coding genes in a genome (known as the exome). It consists of first selecting only the subset of DNA that encodes proteins (known as exons), and then sequencing that DNA using any high throughput DNA sequencing technology. There are 180,000 exons, which constitute about 1% of the human genome, or approximately 30 million base pairs, but mutations in these sequences are much more likely to have severe consequences than in the remaining 99%.[1] The goal of this approach is to identify genetic variation that is responsible for both mendelian and common diseases such as Miller syndrome and Alzheimer’s disease without the high costs associated with whole-genome sequencing.
Prenatal DNA sequencing
About 15% of DNA that circulates in pregnant female derives from the fetus. Shotgun sequencing of father’s genome allows genotyping (along with with fetal blood DNA) of the baby genome. This is done with fairly high accuracy, and is minimally invasive. Can use to figure genetic diseases in fetus.
Genomic DNA footprinting
A method to identify regulatory regions in the genome by measuring the accessibility of chromatin in a given cell type.
The following cleavage agents are described in detail: DNase I is a large protein that functions as a double-strand endonuclease. It binds the minor groove of DNA and cleaves the phosphodiester backbone. It is a good cleavage agent for footprinting because its size makes it easily physically hindered. Thus is more likely to have its action blocked by a bound protein on a DNA sequence. In addition, the DNase I enzyme is easily controlled by adding EDTA to stop the reaction.
Endonuclease cuts DNA based on accessibility, those that are cut and bound often by transcription factors have
been cut by the DNase I.
RNA interference (RNAi)
An endogenous gene silencing mechanism, present in virtually all eukaryotic cells, by which short double-stranded RNA molecules induce translational inhibition
and/or degradation of mRNAs containing partially complementary sequences.
Small interfering RNAs (siRNAs):
22–24‐nucleotide small RNAs that are generated from longer double‐stranded RNA precursors by the ribonuclease Dicer. They can be used to suppress
homology-containing transcripts in a transcriptional and post-transcriptional manner.
Can also target viral RNA.
RNA-induced silencing complex (RISC)
The catalytic effector complex of RNA interference
(RNAi)-mediated gene silencing. The RISC is a multiprotein complex that incorporates one
strand of a small interfering RNA (siRNA) or microRNA.
is a multiprotein complex, specifically a ribonucleoprotein, which incorporates one strand of a double-stranded RNA (dsRNA) fragment, such as small interfering RNA (siRNA) or microRNA (miRNA).[1] The single strand acts as a template for RISC to recognise complementary messenger RNA (mRNA) transcript. Once found, one of the proteins in RISC, called Argonaute, activates and cleaves the mRNA. This process is called RNA interference (RNAi) and it is found in many eukaryotes; it is a key process in gene silencing and defence against viral infections
Argonaute (AGO)
A family of proteins that bind to small RNAs and that are conserved in all domains of life. Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). Small RNAs guide Argonaute proteins to their specific targets through sequence complementarity (base pairing), which then leads to mRNA cleavage or translation inhibition.
PIWI-interacting RNAs (piRNAs)
Small RNAs that are associated with the PIWI protein
complex and that emanated from transposon-like elements
RNA-mediated Transcriptional Gene Silencing (TGS) :
a broad group of RNAi-related mechanisms, where sRNAs silence specific regions of the genome by mediating the establishment of repressive chromatin modifications.
SWI/SNF
SWI/SNF protein has a DNA-stimulated ATPase activity and can destabilize histone-DNA interactions in reconstituted nucleosomes in an ATP-dependent manner, sliding the histone down the DNA chain.
Dicer
Dicer cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short double-stranded RNA fragments called small interfering RNA and microRNA, respectively. These fragments are approximately 20-25 base pairs long with a two-base overhang on the 3’ end. Dicer facilitates the activation of the RNA-induced silencing complex (RISC), which is essential for RNA interference.
DNA clamp
A DNA clamp, also known as a sliding clamp, is a protein fold that serves as a processivity-promoting factor in DNA replication. As a critical component of the DNA polymerase III holoenzyme, the clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand.
Allows DNA polymerase III to move much faster than DNA Polymerase I
How does p53 cause cell cycle arrest?
If there is significant DNA damage, p53 stimulates production of p21. p21 binds and inhibits all cyclin-CDK complexes causing arrest of cell cycle until DNA damage repaired and p21 levels drop. Mutations in p53 are associated with most cancers.
What are the basic amino acids and how do they aid in biological fnx?
The basic amino acids are arginine, lysine, and histidine. Arginine is the most basic.
Arginine and Lysine are critical in Histones, as they add the (+) at the end of the histones, allowing DNA to bind to it.
Which ribosomal site does the initial Met tRNA enter during translation?
The small ribosomal subunit is joint by the large subunit, initiation factors, and the initiator tRNA. The initiator tRNA (Met or fMet) enters the P-site. The initiation factors dissociate from the ribosome-mRNA complex once initiation is complete.
Most important CDKN
Cyclin dependent kinase inhibitors
The Cyclin D-CDK4/6 complex can be inhibited by other proteins. Two important CDK inhibitors are p21 and p16. Loss of p16 function is associated with melanoma.
What cyclin-CDK complex triggers DNA replication?
Cyclin E is degraded allowing for Cyclin A to complex with CDK2 and this complex triggers DNA replication.
CDK4 & CDK6 come prior in G1, will lead to CD2 phosphorylating Rb protein (releasing E2F)
Transcriptional Control Regions
o Gene will be regulated based on DNA sequence that is near or far (can be upstream in the near (-) base pairs)
-Even introns can effect
o Promoters = Transcriptional Control Region
-Includes the TATA box or TATA region (and often a CAAT sequence 25-70 basepairs upstream of AUG start codon)
o Enhancers: Stretch of DNA that increases the rate of transcription when bound by transcription factors
o Silencer: Stretch of DNA that decreases rate of transcription when bound
Disulfide Bridges
the Sulfur on Cysteine amino acids can link with another Cysteine, forms a covalent bond
- Very common in proteins
- Human insulin uses disulfide bridges to link chains
Glycosylation
many secreted or surface cell receptors are glycosylated
- O linked glycosylation: Sugars added to Ser or Thr
- N linked glycosylation: Sugars added to asparagine
Protein Teriary Structure
Overall spatial arrangement of atoms in a polypeptide chain or in a protein
o Two major classes
Fibrous proteins
-typically insoluble; made from a single secondary structure
-Provide a structural basis on body
globular proteins
- water-soluble globular proteins
- lipid-soluble membranous proteins
Importance of turns and loops in protein/tertiary structures
o Glycine and Proline are most often in turns and loops
o Loops can have functions which are not just to do with getting the protein folded up correctly.
-Ex: the loops of the V regions of antibodies bind antigens
Kd=dissociation constant
Kd= [P] [L] / [PL]
X-Ray Crystallography
Purify the protein Crystallize the protein Collect diffraction data Calculate electron density Fit residues into density • Pros: No size limits -Great for large proteins Cons: Crystallization is the rate-limiting step -Very poorly understood process that is difficult -Is a static picture
Biomolecular NMR
Purify the protein (Need in large concentration) Dissolve the protein Collect NMR data Assign NMR signals Calculate the structure o Pros: -No need to crystallize the protein o Cons: -Need very concentrated proteins -Very difficult for large proteins (
Why don’t all proteins refold into their native structures after denaturation?
o In the cell the protein folds as it is synthesized.
o The protein may have been irreversibly insolubilized by the denaturation process.
o Proper folding of some proteins in the cell is aided by various chaperone proteins.
Chaperones and protein folding
o First class of chaperones: Hsp70/Hsp40
-prokaryotic homologs: DnaK and DnaJ
bind to hydrophobic region of unfolded protein and prevent aggregation
-help transport some proteins cross membranes in unfolded states
o DnaK and DnaK
-Prevents aggregation for the hydrophobic regions of unfolding
second class of chaperones: chaperonin
E. coli: GroEL/GroES complex, required for 10-15% of cellular protein folding
In Eurkaryotes 7 subunits that are unique
GroEL belongs to the chaperonin family of molecular chaperones, and is found in a large number of bacteria.[1] It is required for the proper folding of many proteins. To function properly, GroEL requires the lid-like cochaperonin protein complex GroES. In eukaryotes the proteins Hsp60 and Hsp10 are structurally and functionally nearly identical to GroEL and GroES, respectively.
Prolyl isomerase
is an enzyme found in both prokaryotes and eukaryotes that interconverts the cis and trans isomers of peptide bonds with the amino acid proline. Proline has an unusually conformationally restrained peptide bond due to its cyclic structure with its side chain bonded to its secondary amine nitrogen.
Most amino acids have a strong energetic preference for the trans peptide bond conformation due to steric hindrance, but proline’s unusual structure stabilizes the cis form so that both isomers are populated under biologically relevant conditions.
Initiation of Transcription
• Initiation: The Polymerase binds to the Promoter region, is still a “closed complex” as the DNA strand has not open
o Upstream sequences help the polymerase find the promoter
-Initiation Co-Factors: These factors (proteins) help the polymerases find the promoter region
-TBF (TATA box finding protein) Helps the promoter be seen by the RNA Polymerase
o Polymerase melts DNA = creates an “open complex” or the replication bubble
o Begin initial rNTPS (RNA nucleotriphospates) and linking them
C-Terminal Domain CTD of RNA Polymerase II
Binds to the proteins that regulate elongation and processing of the RNA transcript
In humans, the CTD of RNA polymerase II typically consists of up to 52 repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser.[1] This allows other proteins to bind to the C-terminal domain of RNA polymerase in order to activate polymerase activity. These domains then involved in the initiation of DNA transcription
RNA Polymerase II - What does it need to bind?
TATA box is the most typical of promoter regions.
Also needs general transcription factors that will recognize the TATA box (The RNA polymerase cannot find the TATA box on it’s own)
Intron specifics
o At beginning of Intron (5’ end) there is always a G U nucleotide
o At 3’ there is always a A G residue
-There is a branch A in the middle of the structure Adenine
Splicing is done via Lariat Intermediate
o Branch point A of intron will have an hydroxide,
o Than the Hyroxide of 3’ end will attack
This will ultimately detach the intron, allows the exon to come together
Ubiquination
Adding a small regulatory protein of ubiquitin
o Often times ubiquitin is added to proteins to mark it for degradation via proteases
-proteins deemed necessary before cell division, or defective proteins that can’t fold properly
Proteases and peptidases
•hydrolyzes peptide bonds at specific amino acids in the peptide
-Proteases can vary drastically on their mechanisms of cleavage/ hydrolysis
♣ Many are very specific and target only certain molecules
• Ex: Angiotensin converting ezyme ACE
• Ex: Activating digestive enzymes such as trypsin or chymotrypsin
o Example: Blood clotting cascade
-Ultimately the Xa factor will cleave Prothrombin into Thrombin
-Thrombin then cleaves fibrinogen to fibrin
Secondary Structure - Alpha Helix
Alpha Helix: H bonds between the Carbonyl Oxygen and the Amine Hydrogen
o Between backbone carbonyl with Hydrogen residue of a nitrogen backbone (not from R-group of peptides)
Alpha Helices form strong proteins
-Ex: Keratin structure (hair, nails), myosin, fibrinogen
Forms a right hand screw
-H-bonds between CO of residue n and the NH of residue n+4
fit very well into Major Groove of double stranded DNA
Certain amino Acids are good for Alpha Helix formation
-Ala and Leu are Strong Helix formers
Proline and Glycine are Alpha Helix Breakers
Alpha Helix formers and breakers
Certain amino Acids are good for Alpha Helix formation
- Ala and Leu are Strong Helix formers
- Proline and Glycine are Alpha Helix Breakers
Beta Sheets
Beta Sheets Have more extended backbone
Looks like pleated sheets
Hydrogen bonds between neighbors of other sheets
Side chains protrude from the sheet alternating in up and down direction
- Anti-parallel= R-groups pointing in opposite direction
- Parallel= R-groups pointing in the same direction
Peptide Bond
One loses a hydrogen and oxygen from its carboxyl group (COOH) and the other loses a hydrogen from its amino group (NH2). This reaction produces a molecule of water (H2O) and two amino acids joined by a peptide bond (-CO-NH-). The two joined amino acids are called a dipeptide.
The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) refers to the start of a protein or polypeptide terminated by an amino acid with a free amine group (-NH2)
The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH).
Kd= Binding Association
Smaller Kd= better binding affinity so a compound bind better (faster)
What is read depth?
number of times a base in the reference genome was independently sequenced
Competitive and non-competitive inhibitors on Km and Vmax value of enzymes
Competitive Inhibitors–> Will increase the Km (due to competing with substrate for binding spot)
Noncompetitive inhibitors–> Will decrease the Vmax (lower the amount of enzymes available)
What is a usage for CRISPR/Cas9?
The CRISPR/Cas system has been used for gene editing (adding, disrupting or changing the sequence of specific genes) and gene regulation in species throughout the tree of life. By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism’s genome can be relatively cheaply cut at any desired location
What isomers are all amino acids?
The are all L-sided or L isomers
What two amino acids are negatively charged?
Aspartate and glutamate
What amino acid is implicated in scurvy?
hydroxy proline
During O linked glycosylation where are sugars added?
Serine or Thrionine
Replication Protein A (RPA) in Eurkaryotes
Keeps the DNA from winding back on itself, and removes secondary structure
o Keeps the DNA unwound so the Polymerase can replicate
Replication protein A (RPA) is a protein that binds to single-stranded DNA in eukaryotic cells.[1] During DNA replication, RPA prevents single-stranded DNA (ssDNA) from winding back on itself or from forming secondary structures. This keeps DNA unwound for the polymerase to replicate it. RPA also binds to ssDNA during the initial phase of homologous recombination, an important process in DNA repair and prophase I of meiosis.
Telomerase
Able to add RNA template to the end of the chromosome, adding more of the TTAAGGG buggering zone
Cancer cells (& stem cells) have noticeably increased telomerase activity
- It’s in cancer’s best interest to keep its genetic code “young”
- Normal aging cells have inhibited telomerase activity, so the telomere regions shorted with cell “aging”
Point Mutation- Transition
o Purine replaced by Purine, or Pyrimidine replaced by pyrimidine
EX: Adenine replaced by Guanine (both are purines) will change the respective attachement
Point Mutation- Transversion
o Transversion: Purine replaced by pyrimidine or vice-versa
EX: G replaced by a T
Base Excision Repair
o The Glycolase Endonuclease recognizes the damaged base (it recognizes specific mismatched/damaged DNA)
- Hydrolyzes N-glycosidic bond yielding an AP Site
- Glyoclases are designed to recognize specific base pair
o The AP Nuclease will clave the 5’ carbon sugar phosphate backbone
-Different endonuclease cleaves the 3’ carbon sugar
o Polymerase gives appropriate Nucleotides via Polymerases and then sealed by DNA Ligase
BER fixes Types of Damaged Bases: X-rays, Oxygen Radicals, Alkylating Agents, Spontaneous reaction
Nucleotide Excision Repair (NER)
Similar to BER, but replaces much larger strands of nucleotide damage, that WILL cause distortion damage to the overall DNA strand
o More versatile repair- will kick in when DNA strand is causing distortion damage and blocking polymerase function
STEPS of NER
-Multi-protein complex must recognize damaged strand
-Due to strand damage: Must use Helicase to unwind the DNA strand
• Helicase will form small ~25 nucletotide bubble
-Two endonucleases must remove both strands that are within the bubble
-DNA Polymerase replace nucleotides and DNA Ligase repairs the nicks/lesions
NER machinery recognizing Damage based on region
Global Genome NER
• Recognizes damage anywhere in Genome
• Defects cause cancer (Xerodera Pigmentosum)
Transcription Coupled NER
• Recognizes damage within a transcription region
o Where an RNA molecule is stalled due to a lesion
• Cockayne Syndrome
Mismatch Repair (MMR)
Newly synthesized daughter strand is prone to errors, specific MMRs are able to comb through and find errors
o Fxn: It removes Nucleotide from the new daughter strand
Relies on two Protein complexes: MSH and MLH
-“Muts Homologs”= MSH and MLH
Mismatch repair is a highly conserved process from prokaryotes to eukaryotes. The gene products are, therefore, called the “Mut” proteins, and are the major active components of the mismatch repair system. Three of these proteins are essential in detecting the mismatch and directing repair machinery to it: MutS, MutH and MutL (MutS is a homologue of HexA and MutL of HexB).
MutS forms a dimer (MutS2) that recognises the mismatched base on the daughter strand and binds the mutated DNA. The entire MutSHL complex then slides along the DNA in the direction of the mismatch, liberating the strand to be excised as it goes. An exonuclease trails the complex and digests the ss-DNA tail. The exonuclease recruited is dependent on which side of the mismatch MutH incises the strand – 5’ or 3’. If the nick made by MutH is on the 5’ end of the mismatch, either RecJ or ExoVII (both 5’ to 3’ exonucleases) is used. If, however, the nick is on the 3’ end of the mismatch, ExoI (a 3’ to 5’ enzyme) is used.
The entire process ends past the mismatch site - i.e., both the site itself and its surrounding nucleotides are fully excised. The single-strand gap created by the exonuclease can then be repaired by DNA Polymerase III (assisted by single-strand-binding protein), which uses the other strand as a template, and finally sealed by DNA ligase. DNA methylase then rapidly methylates the daughter strand.
o How does MMR recognize nascent (New) strand?
New strand of DNA is recognized by hemimethylation state in E. coli, and by nicks on newly synthesized DNA strand in humans.
In prokaryotes, the new daughter strand is usually not methylated, in gram-negative bacteria, transient hemimethylation distinguishes the strands (the parental is methylated and daughter is not).
Eukaryotes On Nascent (new) lagging strand the nicks from the Okazaki fragments -On Nascent Leading Strand Nicks also on leading strand= ribonucleotides which are involved with the nicks via RNase 2
Trans-lesion Synthesis
Trans-lesion synthesis:
Last resort, used when DNA damage is so severe that DNA replication machinery cannot advance. Special DNA polymerases repair damaged DNA by inserting nucleotide bases, don’t have template but not completely arbitrary. Very mutagenic! (No proofreading)
o DNA is too damaged for BER, NER, and MMR
o “error-pone” or “bypass” Polymerases are used by
o DNA replication complex/machinery is stalled at highly damaged DNA
- The bypass polymerases come and attach to the main DNA replication machinery, allow it to add nucleotides without a template
- Has no proofreading or 3 to 5 exonuclease capabilities
Nonhomologous end-Joining (NEH)
Both strands of DNA are broken, DNA ligase complexes bring the broken helix back together
-Highly mutagenic, very likely to have deletions or insertions= Due to broken helix being brought back together
Ku is a protein that binds to DNA double-strand break ends and is required for the non-homologous end joining (NHEJ) pathway of DNA repair. Ku is evolutionarily conserved from bacteria to humans.
NHEJ initiates with recognition of the DS break by Ku • Ku recruits DNA-dependent protein kinase
NHEJ variably uses combinations of:
• nuclease to remove the damaged DNA if there is damage
(Ku, DNA-PKcs + Artemis complex) • polymerase to fill gaps
Repair finishes with ligase to restore the continuous phosphodiester backbone on both strands (LIG-4)
Homologous recombination
It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks.
-Due to the homologous chromosome available–>Has a template from previous replication available, able to make accurate reattachment of broken-off helix
Single Strand DNA breaks
What protein to the rescue?
o PARP Poly(ADP-ribose) Polymerase enzyme
Activated by single strand break
Will release signals that there is broken single strand of DNA
• Will lead to enrichment of repairing proteins
Thermodynamic Laws
1) Energy is neither created or destroyed
2) Entropy of the Universe is always increasing
deltaG = deltaH - TS
A negative H is heat loss in a system= Desirable because it will lead to a -G
A positive S ultimately will lead to a –G energy (increasing entropy is a desirable part of reaction)
Putting in heat into a system (+H), or Organizing a structure (negative entropy) is not desirable= +G
deltaG = -nFdeltaE
G = -nFE
n= no. electrons transferred, F = Faraday constant,
E= difference in reduction potential in volts
• A increase in electron transfer, or increase in reduction potential= a higher (-)G value, more desirable
A positive E value is the equivalent of a -G, which is desirable
Reduction Potential E - ability of something to accept electrons
- The more (+) E is more likely to accept electrons/ get reduced easier
- The more (-) E is Willing to give away electrons more readily
Expression Vector
plasmid or other piece of genetic information that is being put into a target cell/ a target genome to be expressed
Restriction sites
• are locations in the DNA that are recognized by the Restriction Enzymes, they are typically 4-8 nucleotides long
o They are cut by the restriction enzymes, and sealed back together by DNA ligase
Microarray analysis
o Uses a microarray chip with specific hybridization probes
o Only good for measuring DNA and genes
Terrible with mRNA and with alternative splicing
o Identify similar patterns of gene expression across gene (rows) or microarray experiments (e.g. cell types) columns
Polymerase Chain Reaction
• Used to copy/magnify DNA across a strand, creating thousands or millions of copies of that DNA
o Need a primer for the specific sequence, and a Polymerase from a thermophile
-The famous heat-stable Polymerase is the Taq polymerase
-Important note Initially heat 95 degrees Celsius to met, allows Primer to attach
• Than 72 degrees Celsius for the Taq polymerase
o Thermal Cycling cycles of DNA melting via setting the temp near the Tm, than relying on Taq polymerase or something similar to create the strands (need free dNTPS)
-The DNA that is created via PCR can become the template for the primers and Polymerase
-Selectivity is based on the Primers used on the template strand need to have a good idea of the genome that is undergoing PCR
o In the first step, the two strands of the DNA double helix are physically separated at a high temperature in a process calledDNA melting. In the second step, the temperature is lowered and the two DNA strands becometemplatesfor DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use ofprimersthat arecomplementaryto the DNA region targeted for amplification under specific thermal cycling conditions.
o As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion achain reactionin which the DNA template isexponentiallyamplified. PCR can be extensively modified to perform a wide array ofgenetic manipulations.
Hemoglobin structure
Hemoglobin is the chief O2 binding protein in Red Blood Cells, with two alpha and Beta subunits. Each subunit can bind one oxygen.
Cooperative Binding:
There are two states that the Hemoglobin is in: Taut and Relaxed.
- With no oxygen bound, the hemoglobin is in the Taut phase –> has low 02 affinity
- Once an oxygen binds, the hemoglobin relaxes and has its O2 affinity increase drastically (relaxes state= increase in 02)
In the body: High O2 content in lungs allows the hemoglobin to become saturated.
Low 02 content in peripheral tissues –> decreases binding of O2, and allows the hemoglobin to become taut and release into the tissues.
Next Generation
Optical detection
Illumina- short read sequences “Private Sector”
- More precise work at the short reads
- 50-250 base pair reads (roughly around 100 base-pairs per run)
- Millions to several billions of fragments that consist of roughly 100 base pairs
- Error rate= roughly 1 in a million ( 1*10^-6)
- High read-depth, means that the base pairs have been read many times over, allowing comparison between one another
- Focused on Exome-sequencing
Pacific Biosciences- long read sequences
- Reach around 10,000 sequencing lengths and 10,000 base base pairs
- High error rate (roughly 10%)
Single Nucleotide Variation Identification
- Identical genome sequences are put together, then they are fragmented/ “shotgun” sequencing”
- Now the copied genomes are a bunch of fragments, these fragments are called “sheared random fragments”
With these fragments, they are compared to reference strands. The reference strands are called the “reads”.
Match the fragment strands with the reads to test the fragments for accuracy.
Reads and fragments form contigs together. These contigs can than be compared with the human genome. Human genome project needs to have plenty of read coverage (many reads of human genome).
Adding the Cap to the mRNA
At 5’ of the polypeptide, cut off gamma phosphate with triphosphatase
Than methyl group added via S-adenosylmethione
Guanine 7 methyl transferase adds the 7-methyl guanine
Which U of snRNPs attack what portion of the Introns.
The 5’ splice site is 1st recognized by base pairing to the U1 snRNA.
The branch point is recognized by base pairing to the U2 snRNA.
Splicing of pre-mRNA is probably an RNA catalyzed reaction
Splicesome
The spliceosome is a large ribonucleoprotein complex composed of the pre-mRNA, over 100 proteins,
and 5 small nuclear RNAs (snRNAs)
Proliferating cell nuclear antigen (PCNA)
is a DNA clamp that acts as a processivity factor for DNA polymerase δ in eukaryotic cells and is essential for replication. PCNA is a homotrimer and achieves its processivity by encircling the DNA, where it acts as a scaffold to recruit proteins involved in DNA replication
The encoded protein acts as a homotrimer and helps increase the processivity of leading strand synthesis during DNA replication.
XPB and XPD
XPB is an ATP-dependent DNA helicase in humans that is a part of the TFIIH transcription factor complex (part of RNA Polymerase II).
XPD is a protein involved in transcription-coupled nucleotide excision repair.
Lipid-Soluble Hormones interact with the proteins/transcription factors once they are ligand bound
interact with the proteins/transcription factors once they are ligand bound
oTh e hormone activates them so they can attach to DNA or other proteins
o Examples: Estrogen induces dimerization, activating transcription factors
o Clinical: Tamoxifen
-antagonizes estrogen by binding to ER and preventing recruitment of HAT co-factors
• Limits Breast Cancer’s ability to undergo transcription
Prevents histone acetyltransferase from adding acetyls
Regulating Transcription Factor/Activator entry into Nucleus
o Chaperone proteins and molecules will hold onto the Activator
o Hormone will enter cell and bind to the ligand binding domain of these “chaperones”
-Release transcription factor and allows it to enter cell to manipulate DNA!
• Example: NFkb is chaperoned by lkB
o During inflammation, the lkB will become phosphorylated and ultimately degraded
o This allows NFkB to enter the cell and turn on all the inflammatory immune response (Genes TNF and IL-8)
Releasing neutrophils, machrophages ect.
oExample 2:
NF-ATc is responsible for immune responses = the signal removes the PO3-7
Moves into the nucleus, activates the immune response
Regulating the amount of the transcription factor in the cell
Amount of β-catenin in cell is regulated throughAPC
Beta-Catenin sits in the membrane will build up and eventually make way into the cell membrane as a transcription factor
Eventually will overlfow in and will turn on cell growth genes (can be very cancer related with mutations!!)
Regulating binding to DNA
Id family members negatively regulate DNA binding of bHLH proteins by heterodimerizing through their HLH domains but preventing DNA binding due to their lack of a basic domain.
ID family of proteins: it competes for the e-box, not allowing the Ebox protein to bind and form its diomer
-control when E-box proteins can bind, stops the E-box proteins from activating and forming diomers
E box proteins Bind to specific DNA sites (called e boxes)
- The E-box are genes usually critical for development (muscle development, ect)
Phosphorylation of the DNA-binding protein alters properties
Phosphorylation of CREB promotes transcription by allowing the recruitment of CBP/Pol II
CREB protein the protein is bound already to DNA inactively!!
Sequence of events
A kinase gets phosphorylated, allows it to recognize and interact with the CREB protein
Phosphorylation by Kinase to the CREB, allows the recruitment of CBP/ Pol II will work with DNA
