Term
|
Definition
| DNA sequence that can be transcribed; includes genes for rRNAs, tRNAs, snRNAs, proteins, etc. |
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|
Term
Constitutive Genes
example? |
|
Definition
transcribed at constant level regardless of changes in environment.
Glycolysis Proteins |
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Term
|
Definition
| Transcription changes in response to environment |
|
|
Term
| What is the basis of all geneic information flow in cells? |
|
Definition
| Complimentary base pairing |
|
|
Term
rRNA
Synthetic Capacity, Rate of Degredation, Steady State Level |
|
Definition
58%
Slower than synthesis
83% stable |
|
|
Term
tRNA
Synthetic Capacity
Rate of Degredation
Steady State Level |
|
Definition
|
|
Term
mRNA
Synthetic Capacity
Rate of Degredation
Steady State Level |
|
Definition
32%
Degraded faster than it is made
3% |
|
|
Term
|
Definition
|
|
Term
RNA pol in eukaryotes?
What are they used to make |
|
Definition
multiple RNA pols.
RNA Pol 1: pre-rRNA
RNA Pol 2: pre-mRNA
RNA Pol 3: pre-tRNA |
|
|
Term
| What subunit is important in initiating transcription in prokaryotes? What does it do? |
|
Definition
Sigma factor
Gets the polymerase to the right part to identify where to begin synthesis |
|
|
Term
RNA polymerase mechanism
Where does energy come from? |
|
Definition
Energy comes from Ribonucleoside Tripohs.
-Synthesis only 5' to 3'
-Initiates on dsDNA template to form ssRNA product |
|
|
Term
| Can RNA polymerase proofread? |
|
Definition
|
|
Term
| What strand is the coding strand, anticoding? |
|
Definition
Anticoding=template 3' to 5' (make RNA from this)
Coding= 5' to 3'. It should be identical to synthesized RNA except U,T |
|
|
Term
| Where does transcription begin? |
|
Definition
|
|
Term
| Prokaryoteic Transcription |
|
Definition
| -Prokaryotic promotor("-10" and "-35") upstream of +1 and bind to RNA pol sigma factor. |
|
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Term
|
Definition
| The nucleotide that occurs most often at that given position. |
|
|
Term
| The concensus sequence -35 and -10 of many gense. There is another gene were none of the nucleotides are equal. We are told that they are transcribed efficiently. What might be going on? |
|
Definition
| It is using a different consensus sequence and recognized by a different sigma factor. |
|
|
Term
| Transcription elongation in prokaryotes: |
|
Definition
-RNA pol unwinds the DNA and a transcription bubble formed
-This opens the complex and RNA is slowly formed
-The sigma factors resists RNA pol resists RNA pol moving
-After short RNA is made, sigma dissociates and RNA pol clears from promoter and makes RNA faster. |
|
|
Term
| Transcription Termination (pro) |
|
Definition
| Usually occur at specific 'pause sites' that are often DNA, G:C rich. |
|
|
Term
Where do eukaryotic RNA pols bind?
What do they use? |
|
Definition
| NOT to DNA on their own. They use Transcription factors (TF) to recognize DNA sequences. |
|
|
Term
|
Definition
Complex of proteins forms from binding of TF's
Additional TF's are recruited.
Finally RNA pol 2 initiates and transcripts at +1 |
|
|
Term
3 ways to weaken Histone-DNA binding
What does this do? |
|
Definition
Makes RNA pol/TF more assessable to DNA
-Histone Acetyl Transferases (HATS)
-Histone methylases (methylated)
-Histone Kinases phosphorylate (serine)
Lys and arg for the first 2 are the molecules acetylated and methylated |
|
|
Term
3 ways to increase Histone-DNA binding.
What does this do? |
|
Definition
Lessens the space for Pol/TF to access DNA
-Histone deacetylase (HDAC)
Histone demethylase
Histone phosphatases |
|
|
Term
| Where can gene expression be regulated? |
|
Definition
Many levels:
-Transcription
-mRNA processing/transport
-Translation
-Post translation |
|
|
Term
|
Definition
| Inhibit transcription when bound to DNA |
|
|
Term
|
Definition
| Stimulate transcription when bound to DNA |
|
|
Term
|
Definition
Modulate repressors and activators binding DNA
Small molecules that bind proteins |
|
|
Term
|
Definition
Inducers (inactivate repressors)
Corepressors (activate repressors) |
|
|
Term
| Regulation by Lac operon: |
|
Definition
| Repression and Activation |
|
|
Term
|
Definition
| Contains genes from metabolizing lactorse. Transcribed ONLY when lactose is abunant and glucose is limiting. |
|
|
Term
|
Definition
Long piece of mRNA that have different regions that code for different proteins.
Only in prokaryotes |
|
|
Term
| What is usually bound to either lactose or operator in the Lac operon? |
|
Definition
|
|
Term
| Is lac operon constitutive or inducable? |
|
Definition
| Constitutive. Always synthesized at a low level in prokaryotes. |
|
|
Term
|
Definition
| Repressor binds lactose. Operator is free. RNA pol binds the promotor. More transcription and polysictronic mRNA |
|
|
Term
|
Definition
| Repressor binds to operator. Less RNA pol can bind to the promotor. Low transcription. |
|
|
Term
| What does the polycistronic mRNA in lac operon eventually produce? |
|
Definition
B-galactosidase
Permease
Transacetylase |
|
|
Term
|
Definition
(Also known as catabolite activator protein CAP)
Active when CAMP is abundant.
When this complex CRP*CAMP binds to DNA increase affinity for RNA [pol. greater transcription. |
|
|
Term
| Low Glucose, High Glucose and CAMP |
|
Definition
Low glucose= High Camp. High transcription
High glucose=Low CAMP. Low transcription. |
|
|
Term
|
Definition
| Ability of glucose to reduce cAMP and reduce transcription of multiple genes |
|
|
Term
| If there is a gene____ and its transcription is reduce when glucose levels are high in prokaryotic cell and transcription is elevated when glucose levels are low, what is likey to be regulating the gene? |
|
Definition
| CRP-cAMP colmplex. Complex binds near the promoter of then gene when glucose levels are low. |
|
|
Term
| When targeting enzymes and pathways for therapeutic purposes, what kind of compound does one want? |
|
Definition
| One that inhibits an essential step unique to the offending organism/cell with minimal side effects on human host. |
|
|
Term
| Mechanisms of action for therapeutic destruction of enzymes |
|
Definition
1. Competative inhibition
2. Suicide inactivation
3. Modified substrates that halt many steps in a multi-step process (ex. DNA rep) |
|
|
Term
| Antiviral Drugs Discussed: |
|
Definition
Acyclovinr (acycloguanosine)
AZT |
|
|
Term
Acyclovir is what kind of analog?
What is it missing? |
|
Definition
Purine analog of guanine
3' OH is missing |
|
|
Term
| First step in in bioactivation of acyclovir? What does this do for the drug? |
|
Definition
| Acyclovir monophosphate by HSV kinase (phosphorylated). This gives the drug specificity to infect only cells infected with the virus. |
|
|
Term
| How is bioactivation of acyclovir completed? |
|
Definition
| Forms acyclovir triphosphate via host cell enzymes. |
|
|
Term
| What is acyclovir PPP used for? |
|
Definition
An alternate substrate for HSV-DNA polymerase, substituting for GTP.
When it is incorported into growing DNA chain termination occurs b/c lack of 3' OH |
|
|
Term
| Can acyclovir get into normal cells? |
|
Definition
| Yes, but its not activated unless it has been activated by HSV kinase. |
|
|
Term
|
Definition
|
|
Term
| What is the target of AZT? |
|
Definition
| HIV DNA polymerase (reverse transcriptase) |
|
|
Term
| What type of analog is AZT? |
|
Definition
| Pyrmidine analog with azido group instead of 3' OH group |
|
|
Term
|
Definition
AZT triphosphate by host cell enzymes.
No specificity for HIV enzyme in this step |
|
|
Term
|
Definition
| It is used as an alternate substrate by HIV DNA Pol known more commonly as RT. When used with causes HIV DNA chain termination. No HIV DNA is made from teh HIV RNA to integrate into the genome, so infection ends. |
|
|
Term
| Reasons AZT selects HIV RT DNA synthesis even though host cell enzymes are used? |
|
Definition
-RT has much lower substrate specificity than host DNA polymerases.
-RT more readily incorporates errors into viral DNA.
-HIV RT lacks proofreasing,mistakes remain
Result in a lower fidelity for DNA synthesis than the good host. |
|
|
Term
| What often happens with AZT to decrease its efficacy. |
|
Definition
| Development of resistance to AZT and other anti-HIV drugs is common due to the high mutation rate of HIV RT when DNA is made. This is why drug combos are used. |
|
|
Term
|
Definition
| Methotrexate, 5- flourouracil |
|
|
Term
|
Definition
Key synthetic step with cofactor recycling in DNA dTMP biosynthesis.
Targeting dTMP focuses on DNA, not RNA |
|
|
Term
| What kind of inhibitor is methotrexate? |
|
Definition
| Competative inhibitor of dihydrofolate redcutase |
|
|
Term
| What is methotrexate an analog of? |
|
Definition
|
|
Term
| Essentially, what does methotrexate do? |
|
Definition
| Slows the recycling of the esential methylene tetrahydrate cofactor for thymidallate synthase, decreasing deoxy TMP synthesis. This also decreases DNA replication. |
|
|
Term
| What is 5-flourouracil an analog of? |
|
Definition
|
|
Term
| What kind of inhibitor is 5-flourouracil? |
|
Definition
| suicide inactivator of thymidylate synthase. |
|
|
Term
| What cofactor does 5-flourouracil require? |
|
Definition
| Co-factor thymidylate synthase methylaene-THF (CH2-THF) |
|
|
Term
| What is 5-flourouracil bioactivated to? |
|
Definition
F-DUMP after enters the cell using host cell enzymes.
This is an example of the salvage pathway. |
|
|
Term
| Side effects of chemo, why? |
|
Definition
Hair loss, Intestinal upset (mucosa), Anemia, decreased immunity, tired (bone marrow); on chemo/then off, then on (etc.)
THis is because all of these cells (hair follicles, intestinal mucosa, bone marrow are rapidly dividing. |
|
|
Term
| By the end of 2009, what was the estimation of the total number of HIV infections world-wide, and what area of the world had by far the most? |
|
Definition
33.3 million
Subsaharan Africa |
|
|
Term
| • Name the three key viral enzymes important in HIV viral replication and maturation. What is the major target cell that the virus infects? |
|
Definition
Reverse Transcriptase, Integrase, Protease
Major target cell are those that contain CD4+ on the surface (macrophage, B, T cells) |
|
|
Term
| • Explain the mechanism by which an HIV particle binds and attaches to a host cell just prior to membrane fusion and cell entering. Include discussion of gp160 components and their roles. What are CD4 and CXCR4, and how do they function in this process? |
|
Definition
| • The HIV particle bind with the CD4 particle on the T cell. This happens by a complex known as the gp160 complex. This complex contains a gp 120 and which directly attaches to the CD4 complex, and a gp41 complex which is eventually inserted into the cell membrane. First, the gp120 attaches to the cd4 complex. Next, the complex folds and binds to the chemochine receptor CXCR4 (which is part of the host cell). Next, the gp-41 parti cle is now exposed and inserts HIV through the cell membrane and attaches through the outer surface. |
|
|
Term
| • Review the steps in the process of viral replication starting with the HIV RNA that has entered a CD4+ T cell host. Cite the key enzymes involved from Question 3 above. |
|
Definition
| The HIV RNA has entered the cell and reverse transcriptase first acts on it to form cDNA. Next,integrase acts on the cDNA to integrate the viral DNA into the host cell genome. Now, a provirus has ben formed, this remains silent until this T cell becomes activated. Next, the provirus is transcripted and then spliced and translated. Viral RNA is transported into the cytoplasm where proteins are translated and assembled into virus particles that bud from the cell. This is the immature virus. The mature virus is formed by protease action. |
|
|
Term
| Slide 10: What makes HIV unique among viruses, and what is the reason for this? |
|
Definition
| Our bodies initially produce an immune response to the HIV virus. However, this response does not last. |
|
|
Term
| Four main stages of HIV progression |
|
Definition
Acute Phase
Asymptomatic Phase
Symptomatic Phase
AIDS |
|
|
Term
|
Definition
2-6 weeks
Flu like symptoms
Rapid decrease in immunity |
|
|
Term
|
Definition
Average about 10 years
CD4 numbers remain > 500
Reduce gradually |
|
|
Term
|
Definition
2-3 years
CD4<500
Begin to show more symptoms (increase in amount of virus, decreases immunity) |
|
|
Term
|
Definition
|
|
Term
| What actually causes the death of an aids patient? |
|
Definition
Not the virus itself.
The patient has a very low immune system and many opportunistic virus' and bacteria infections cause actual death |
|
|
Term
| Three cheif characteristics of AIDS |
|
Definition
-Decrease level of CD4 (below 200)
-Increase of aggressive form of Kaposi’s sarcoma or B cell lymphoma
-Increase in susceptibility to infection with opportunistic pathogens. |
|
|
Term
|
Definition
A- Asymptomatic infection
B-Symtomatic condition – start to treat symptoms here
C- AIDS (CD4<200 or <14%) |
|
|
Term
| Which AIDS symptom usually ends the life of a person with this disease? |
|
Definition
|
|
Term
|
Definition
| Highly Active Antiviral Reactive Therapy |
|
|
Term
| What do HAART drugs contain? |
|
Definition
| Combination of drugs with RT inhibitors and protease inhibitors |
|
|
Term
|
Definition
Decreasing viral load.
Increasing CD4 |
|
|
Term
| Major problem with HAART? |
|
Definition
| Difficulty with patient compliance |
|
|
Term
| What question cannot be answered about HAART at this time? |
|
Definition
| Whether the patient can every safely terminate treatment. |
|
|
Term
| What are the access to treatment issues of HAART? |
|
Definition
-Access in third world countries
-Access in poor inner cities and rural America
-Too expensive, esp in third world countires. |
|
|
Term
| Over time, what does HAART lead to? How is this combated? |
|
Definition
| Drug resistance. This is combated by exchanging the drugs used for different ones. |
|
|
Term
| What happens after about 3 months of being treated with HAART agents? |
|
Definition
| Over time, the amount of virus in plasma decrease and is usually at 10 at 3 months treatment. (limit of detection is decreased) |
|
|
Term
| All procaryotic and eukaryotic RNAs are modified and processed except what? |
|
Definition
| Prokaryotic mRNA, it immediately is acted upon by the ribosome. |
|
|
Term
| 3 general types of processing? |
|
Definition
1. Removal of nucleotides
2. Covalent modification of nucleotides
3. Addition of nucleotides not encoded in DNA |
|
|
Term
| How does tRNA processing occur? |
|
Definition
By the 3 main ways of processing.
They are highly processed
-Remove excess nucleotides (endo/exonucleases)
-Addition of nucleotides (all tRNA's have CCA on 3' end)
-Extensive modiications |
|
|
Term
|
Definition
|
|
Term
| How does rRNA processing occur? |
|
Definition
rRNA's are a part of a large transcript.
Many RNases remove excess RNAs->rRNAs |
|
|
Term
Genes for rRNA.
How many.
Euk/Pro? |
|
Definition
| Many genes in eukaryotes and prokaryotes |
|
|
Term
| Where is rRNA made in eukaryotes? |
|
Definition
|
|
Term
| Where do ribosomal proteins bind, what does this do? |
|
Definition
| They bind to the primary transcript and aid processing and prevent degredation. |
|
|
Term
| What is added to eukaryotic mRNA as they undergo extensive processing? |
|
Definition
-CAP @5' END
.N-7 methyl guanine attached to +1 by PDE bond
.2 OH methylation
3' Poly A tail
. |
|
|
Term
| Functions of the mRNA cap |
|
Definition
-Increase stability
-Recognized by splicosomes
-Recognized by ribosomes |
|
|
Term
| What RNA pol is capping associated with? |
|
Definition
| RNA pol 2, b/c form on mRNA (which is RNA pol2) |
|
|
Term
|
Definition
|
|
Term
|
Definition
Cleavage and polyadenylation specificity factor
-Binds to consensus sequence and forms a complex containing RNA and endonuclease.
-Endonuclease catalyses cleavage of the transcript downstream of the polyadenylation forming a new 3' end. -Poly A polymerase binds and makes tail! |
|
|
Term
| What is template independent RNA polymerase? |
|
Definition
|
|
Term
| Functions of Poly A tail: |
|
Definition
Increase stability
mRNA transport from nucleus->Cytoplasm |
|
|
Term
| What does splicing remove? |
|
Definition
|
|
Term
| What does mature mRNA contain? |
|
Definition
|
|
Term
| What happens once mature mRNA is formed? |
|
Definition
|
|
Term
| Where does coupled trancription/translation occur? |
|
Definition
|
|
Term
Splisosomes
What do they do?
What are they made of? |
|
Definition
Splicing
Somplexes of snRNPS |
|
|
Term
|
Definition
| Small RNA's with proteins. |
|
|
Term
| How do snRNPs assemple? What does this form. |
|
Definition
| Sequentially @ each intron, from a sliceosome. |
|
|
Term
| Alternate or differential hnRNA splicing? |
|
Definition
Joins different exons
Form different mRNA
Form different proteins.
(from same inital transcript)
Initally believed rare, now not. |
|
|
Term
| Summary of Euk mRNA processing: |
|
Definition
1. RNA pol 2
2. Cap added
3. Splicing
4. Poly A tail added |
|
|
Term
|
Definition
| Taking nucleic acids and forming the correct amino acid structure->proteins. |
|
|
Term
|
Definition
| Specific nucleotide sequence in mRNA or DNA designate specific AA |
|
|
Term
| What are adapter molecules in translation? |
|
Definition
|
|
Term
|
Definition
-Non overlapping codons
-Proper reading frame
-Univeral
-Unambiguous
-Degenerate or synomous codons (many codons=same AA) |
|
|
Term
How many Stop codons?
How many initiation codons? |
|
Definition
Stop-3
Start-1 (AUG or methionine) |
|
|
Term
| What is the role of tRNA's in translation? |
|
Definition
| convert/adapt mRNA sequence to AA sequence |
|
|
Term
| Characteristics tRNA's use to adapt? |
|
Definition
-Size/Shape (all similar)
-Anticodon
-Acceptor stem |
|
|
Term
| What do amino acyl tRNA's do? |
|
Definition
| Translate mRNA codon sequences to an AA sequence. |
|
|
Term
|
Definition
| Some tRNAs recognize multip codons via imperfect base pairing. |
|
|
Term
| What do we need to know for wobble? |
|
Definition
U is messed up
Binds A or G |
|
|
Term
| What do amino acyl TRNA synthetases do? |
|
Definition
| Join specific AA to appropraite tRNA's to form aminoacyl tRNA |
|
|
Term
| what is special about the amino acid-trna bond? |
|
Definition
| It is a high energy bond that forms the peptide bond during translation. |
|
|
Term
| Can aminoacyl-tRNA's proofread? |
|
Definition
|
|
Term
| Steps in translation (simple) |
|
Definition
-Activation
-Initiation
-Elongation
-Termination |
|
|
Term
| Activation of translation |
|
Definition
| aminoacyl-tRNA synthetases join proper AA with tRNA (covalently bound forms a high energy bond) |
|
|
Term
|
Definition
Large complex of rRNA and proteins that coordinate translation
2 subunits
-small (1rRNA) +proteins (cleft where large sits)
-large (2,3 RNA) + proteins (tunnel to accomodate growing protein) |
|
|
Term
|
Definition
A-Aminoacyl tRNA
P-Peptidyl tRNA
E-Empty tRNA |
|
|
Term
| What does the ribosome accomodate? |
|
Definition
| mRNA, aminoacyl tRNA, peptidyl tRNA, and growing protein |
|
|
Term
|
Definition
N-formyl methionyl tRNA is only in prokaryotic initiation.
Formed by prokaryotic methionyl synthetase and formyltransferases. |
|
|
Term
| Prokaryotic Translation initiation: |
|
Definition
tRNA i/f met is used.
Shine dalgarco sequence in mRNA precedes AUG to determine proper AUG |
|
|
Term
| Prokaryotic initiation factors 1,2,3 |
|
Definition
1 and 3 binds to the small ribosome and prevent the large ribosome from prematurely adding.
2 + GTP binds with the formyl AUG. This interacts and recognizes the S.D.
Large subunit adds. (1,3 released) |
|
|
Term
| Eukaryotic Initiation of translation: |
|
Definition
-eIF-4 binds with the cap and begins formation of the initiation process
-Complex moves along mRNA in the 5 to 3 direction until reaches the AUG. Here eIF-2 brings to small ribosomal subunit.No SD.
-Preinitiation complex pauses and large ribosomal subunit joins with Met-tRNA (this is positioned in ribosome P site)
then the eIF's dissociate. |
|
|
Term
| What is the role of EF-TU GTP in the elongation phase of translation? |
|
Definition
Binds aminoacyl-trna and brings to the A site.
They bind to the acceptor stem of amino-acyl trnas. |
|
|
Term
| Why do EF-Tu NOT bind f-Met-trna? |
|
Definition
| These are for initation. They have a unique acceptor them that prevents them from binding to Ef-TU |
|
|
Term
| Where do the EF-TU GTP aminoacyl tRNA complexes go once formed? |
|
Definition
|
|
Term
| What happens in the elongation phase once the EF-TU GTP touches the ribosome? |
|
Definition
| GTP goes to GDP (high energy bond) and the aminoacyl trana is released into the A site and EF*TU*GDP dissociates |
|
|
Term
Peptidyl transferase?
What. Where. When. |
|
Definition
| Forms the peptide bond in the large ribosomal subunit after dissociation of EF-TU*GDP. |
|
|
Term
| Why peptidyl transferase activity happen? |
|
Definition
| Due to rRNA and ribosomal proteins b/c rRNA has catalytic activity. |
|
|
Term
|
Definition
EF-G*GTP binds ribosome. Moves to put next codon in the open A site; uncharged tRNA in E dissociated (GTP->GDP)
Basically just moving the product from A to P and E... |
|
|
Term
| Termination of translation |
|
Definition
Release factors 1,2,3
Stop codon in the A site.
Ribosome stalls and RF-1 or 2 binds with RF3*GTP which bind the stop codon.
This results in hydrolysis of peptidyl tRNA. Protein released. |
|
|
Term
Coupled transcription and translation.
Process.
What cells? |
|
Definition
ONLY prokaryotes
Prokaryotic mRNA emerges from RNA pol (no processing), ribosomes form initiation complexes. Translation begins before transcription complete.
ONly in pro b/c euk are in different compartments. |
|
|
Term
|
Definition
| Many ribosomes translating proteins on a single mRNA both in prokaryotes and eukaryotes. |
|
|
Term
|
Definition
Binds small ribosomal protein
Inhibits initiation |
|
|
Term
|
Definition
Inhibits peptidyl transferase in large subunit.
Peptide bond formation |
|
|
Term
|
Definition
|
|
Term
|
Definition
| Binds small ribosomal subunit, preventing binding of aminoacyl trna into A site. |
|
|
Term
| Where is a not wiedely used place to regulate gene expression? |
|
Definition
|
|
Term
|
Definition
|
|
Term
| What does heme do, indirectly? |
|
Definition
| Regulates activity of eIF-2 |
|
|
Term
HCI
What does it do. What is the result |
|
Definition
Heme controlled inhibitor.
Phosphorylates eIF2
Global inhibition of translation |
|
|
Term
|
Definition
|
|
Term
| What regulates the trp operon? |
|
Definition
| Repression and attenuation |
|
|
Term
| What is attenuation. Where does it occur? |
|
Definition
ONLY in prokaryotes. For coupled transcription and translation.
-Independent mechanism of sensing trp level. |
|
|
Term
|
Definition
| codes 5 protesin needed to make trp with is required for translation |
|
|
Term
| If TRP is high, what is the activity of the trp operon (repression) |
|
Definition
THe trp operon is low, repressed.
TRP is a co-repressor |
|
|
Term
| If TRP is low, what is the effect on the operon? |
|
Definition
| TRp does not bind to the repressor. operon makes trp |
|
|
Term
| How does attenuation work in the trp operon? |
|
Definition
| Leader region with coupled transcription and translation controls RNA pol moving into trpE gene via 2 trp codoings and 4 GC rich regions. |
|
|
Term
| Formation of leader mRNA in trp operon regulation |
|
Definition
1-2 bound; 3-4 bound
Termination signal
RNA pol leaves before transcribing genes.
2-3 Bound- no pause/termination signal
RNA pol transcribles trp genes.which make proteins that make trp. |
|
|
Term
| Trp is abundant attenuation |
|
Definition
Trp is high. so 1:2 3:4.
RNAP disoccaites. No transcription |
|
|
Term
| Trp is limiting attenutaiton |
|
Definition
| 2:3 and ribosome remains stalled. Termination signal not formed.Trp eventually made |
|
|
Term
| Trp is limiting attenutaiton |
|
Definition
| 2:3 and ribosome remains stalled. Termination signal not formed.Trp eventually made |
|
|
Term
| Free ribosomal proteins translate what proteins? |
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Definition
| Cytoplasmic, Nuclear,Mitochondrial |
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Term
| RER ribosomes make proteins that go where? |
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Definition
| Secreted proteins, PM proteins, ER, lysosomal proteins |
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Term
|
Definition
Attached to ER
Enter ER lumen
Transfer vesicles
Golgi (->lysosome or)
Secretory vesicle
PM or secreted |
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|
Term
| What type of cells do signal hypothesis occur in? |
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Definition
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|
Term
What is a signal peptide?
What type of proteins are they found in? |
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Definition
| 15-30 N terminal AA righ in hydrophobic AA that cause ribosomes to go into ERE; inly present in secreted, PM, lysosome or peroxisome proteins |
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Term
| When is the signal peptide removed? |
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Definition
|
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Term
|
Definition
| Signal recognition particle. Recognized and binds signal peptide as it emerges from ribosome. |
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Term
| What binds the signal peptide? What does it do? |
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Definition
Signal recognition particle binds the signal peptide
Translation now stops |
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|
Term
| What happens to the SRP-ribosome complex in in the signal hypothesis? |
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Definition
| Bound to the ER membrane. |
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Term
| In the signal hypothesis how is the ribosome anchored, and where is it anchored to? What happens next? |
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Definition
Anchored to the Er pore by robophorin/translocon.
Next, SRP is released and signal peptide inserted into pore. |
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Term
| Role of signal peptidase in signal hypothesis |
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Definition
|
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Term
| Role of signal peptidase in signal hypothesis |
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Definition
|
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Term
| How are proteins modified within the ER? |
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Definition
| Transport vesicles, golgi, secretory vesicles. |
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Term
| What proteins are normally glycosylated? |
|
Definition
| Most secreted and membrane bound proteins |
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|
Term
| What is dolichol phosphate? Found? What cells? What does it do? |
|
Definition
Sugar.
Found in ER membrane of eukaryotes
Differnet carb bodies add to dolichol phosphate |
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Term
|
Definition
| Removes pre formed block of sugars |
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Term
| Proteins that end up in Lysosomes are modified in ER or Golgi by addition of ? |
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Definition
|
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Term
|
Definition
| Importin complexes. Protein complex enters. Like Yon! |
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Term
|
Definition
| combining DNA from different sources |
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Term
| Steps in recombinant DNA formtion |
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Definition
1. Isolatte and purify vector and target DNA
2. Cleave vector and target DNA @ specific sequences via restriction endonucleses
3. Ligate target DNAs into vector recombinant DNA
4. Introduce recombinant DNA into appropriate host (Bacteria yeast, mammal, embryo)
5.Replication/Expression of recmobinant DNA in host cells
6. Identify host cells that specifically contain DNA of interest via screening or selection. |
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Term
|
Definition
| Carrier DNA into which target DNA is inserted: often plamids |
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Term
|
Definition
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|
Term
| What is the purpose of the origin of replicaiton? |
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Definition
| ORI present so it can be replicated in the host. |
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|
Term
| What is a shuttle vector? |
|
Definition
| Ability to be used in both bacteria hosts and eukaryotic cell hosts. |
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|
Term
| What do restriction endonucleases do? |
|
Definition
| Generate sticky complimentary ends on target DNA and on vectors, allowing insertion of target into a specific site of vector. |
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Term
|
Definition
Plasmids constructed to contain this. Forms beta galactosidase
B galactosidase hydrolyzes X gal to blue |
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|
Term
| What do PVU I and PSTI do to Lac z? |
|
Definition
| Make it from white colongies |
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Term
|
Definition
| Collection of host cells with recombinant vectors that contain inserts that gother repesent all of the DNA in an organisms genome. |
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Term
|
Definition
| Recombinant DNA libraries that represent all of the mRNAs transcribed in a cell, tissue or organism under certain conditions. cDNA = complimentary, dS dnA |
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Term
|
Definition
-Isolate eukaryotic mRNA on oligo dT columns via poly A tail
-mix oligo dT and bind poly A and acts as a primer for RT
-RT makes cDNA from mRNA template
-RNase H partially degrades mRNA ->multiple RNA primers fro DNA pol 1 synthesizes complimentary DNA as degrades RNA primers
-DNA ligase joins DNA fragments to dsDNA |
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|
Term
|
Definition
-Scren or proble clones to determine which contains insert of intrest
-Probes are molecules that specifically recognize DNA of intrest |
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|
Term
| Process of screening libraries |
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Definition
Spread cells from library on plate
Blot some cells in each colong onto filters
Denature DNA from each colony on filter
Reach with probe containing DNA sequence complimentary to DNA of interest
Probe specifically hybridizes with DNA of intrest |
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|
Term
| Prokaryotic expression vectors: |
|
Definition
Addition to marker gene and ori
-multiple cloning site preceded by strong promoter
-Target DNA inserted in site, after ATG, which is preceded by Shine delgarno sequence
-Transcription Translation Signal
-Protein has a few N terminal amino acids not present on native protein
Human: growth hormone, insulin, blood coagulation factors, |
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|
Term
| Where are many eukaryotic proteins also expressed? |
|
Definition
| Prokaryotes. They aren't active b.c prokaryotes lack post translational modification enzymes. |
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Term
Hosts for eukaryote expression?
What can they do? |
|
Definition
Yeast, cultured cells.
Can splice introns
Insert genomic or cDNA |
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Term
|
Definition
| Recombinant DNA introduced into zygote or early embryo. All cells in offspring contain recombinant DNA |
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|
Term
| Applications of recombinant technology in prokaryotes? |
|
Definition
Genomes of many bacteria have been sequenced. Better understanding of microbial bioogy and virulence factors; may aid development of new drugs to treat/prevent human diseases.
--especially important now bacterial resistance |
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Term
| Applications of human treatment in recombinant technology? |
|
Definition
Human proteins. Various diseases.
Proteins from bacteria.
Human gene therapy
STEM CELL RESEARCH. restrictions |
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|
Term
| Diagnosis of human diseases using recombinant DNA technology? |
|
Definition
Genetic diseases. Coagulation factors. Lesch Nyhan Syndrome. Sickle Cell. Thalassemias.
RFLP |
|
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Term
|
Definition
Restriction fragment lenght polymosphism
Follow patterns of disease inheritance as a family, and diganose and counsel prospective patients.
Also, differentiate different people from milions of others. |
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|
Term
| Polymerase Chain Rxn (PCR) |
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Definition
| Procedure quickly amplifies select DNA sequences from very small amount of DNA sample. |
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Term
| What must you know in PCR? Why? |
|
Definition
| Sequence of DNA bounding region of interest to construct approprate primers |
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|
Term
| What do you do with the DNA in PCR? |
|
Definition
Place in tube with primers, heat stable DNA pol, and 4DNTPs.
Tube sealed and placed in a thermocycler programmed to heat for a specific time, cool, repeat. |
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Term
|
Definition
Amplify genes w/o cloning
test or diagnose genetic diseases amniotic
Find bacterial diseases before widesread
Monitor cancer chemotherapy
Forensics
Moleculer evolution |
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