Important Vocab First M2M exam Flashcards
(101 cards)
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.
