Biochem: DNA, RNA and their friends
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| Define the term supercoiling | DNA double helix that have additional twists applied to them that have coiled up upon themselves
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| What is a purine? | – heterocyclic compound consisting of pyrimidine fused to a 5 member imidazole ring (adenine and guanine)
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| What is a Pyrimidine? | hererocyclic compounds with nitrogen atoms at positions 1 and 3 of a 6 member ring (thymine, cytosine, uracil)
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| What is a Deoxyribonucleosides? | consist of a nitrogenous base and a deoxyribose sugar (H present at 2’ C)
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| What is a Ribonucleotide? | nitrogenous base (attached to 1’ C by B-N-glycosidic bond), a ribose sugar (OH at 2’ C), and a phosphate group (backbone of DNA, attached to 5’ C) – present in RNA molecules
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| What is a Deoxyribonucleotide? | nitrogenous base, deoxyribose sugar and a phosphate group (present in DNA molecules)
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| Describe the phosphodiester bond | The phosphodiester bond is between the 3’ OH group of the deoxyribose/ribose on the last nucleotide and the 5’-phosphate of the dNTP/NTP precurson – basically it is a bond that connects nucleotides in DNA
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| What is the function of modification made to tRNA and rRNA for (ie. 4-thiouracil, dihyrdouracil)? | enzyme/tRNA recognition, tRNA condon recognition, peptide synthesis by ribosome
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| What is the difference between a ribose and deoxyribose? | ribose: OH on 2'carbon
Deoxyribose: -H on 2'carbon
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| Difference between nucleoside and nucleotide? | Nucleoside: nitrogenous base and a ribose/deoxyribose sugar
Nucleotide: nitrogenase base, ribose/deoxyribose and a phosphate grp
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| How do you build DNA or RNA polymers | DNA/RNA poly catalyzes the formation of a phosphodiester bond between the 3'OH of deoxyribose/ribose on the last nucleotide and the 5'-phosphate of the dNTP/NTP precursor
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| What is the ratio of purines to pyrimidines in double stranded DNA? | 1:1 (ie. 50% purines and 50% pyrimidines)
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| What is Chargaff's rule? | A=T and C=G
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| What are the 7 properties of DNA? | 1. Two polynuc right handed double helix
2. Antiparallel.
3. sugar-phosphate outside, bases inside
4. bases bound by H-bonds
5. complementary base pairing
6. bases .34nm apart, 10 bases/turn,external diameter is 2nm
7. major and minor groove
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| How long is each DNA stand/cell? | 2 metres
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| What are the different structural forms of DNA? | Z-DNA, B-DNA and A-DNA
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| Describe Z-DNA | left handed helix. may be involved in gene regulation
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| Describe A-DNA | right handed helix, dehydrated state, no biological significance.
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| In nature how does DNA exist? | It exists in tertiary (supercoiling) and quarternary structures (coiling with histones).
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| What protein is involved in creating supercoiling? | topoisomerase
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| Describe structures and roles of the mRNA | linear and single stranded, carries codon information for translation (2%)
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| Describe structures and roles of the tRNA | folded loop structures formed by intra-molecular H-bonding, carries amino acids to ribosomes (16%)
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| Describe structures and roles of the tRNA | linear, single stranded, folded molecule that complexes with protein to form ribosomes (82%)
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| What is Tm? | the temperature at which 50% of DNA becomes single stranded.
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| What influences Tm? | high C-G content (incr Tm), high salt concentration (incr Tm)
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| What affects the rate of annealing of DNA strands? | Size of DNA (long DNA takes longer), complexity of DNA
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| Describe the function of microRNA? | regulate gene expression by binding to a complementary single stranded RNA which triggers the cell to destroy it
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| Describe FISH. | Take a single stranded DNA with gene sequence of interest with a fluorescent probe let it anneal to DNA in cell and look for fluorescence.
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| How do Nucleoside analog drugs inhibit DNA replication? | analogs have a higher affinity for reverse transcriptase and incorporate the analog without an OH grounp on the 3' carbon.
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| What group does didanosine have in its 3' carbon slot? | doesn't have one
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| What analogs are used for HIV treatment? | Didanosine, tenofovir
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| What analogs are used to kill rapidly dividing cancer cells? | Cytosine arabinoside (cytarabine) and Vidarabine (adenosine arabinoside)
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| How does vidarabine and cytarabine inhibit the activity of DNA poly? | 3' OH is in a different planar configuration
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| What happens to DNA with a lot of methyl groups? | DNA transcription is shut of.
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| What is the purpose of azacytidine and 5-azacytidine? | Used to treat cancer by inhibiting methyltransferase enzymes from adding a methyl group to them.
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| Define supercoiling. | Supercoiling compacts DNA; negative supercoiling promotes strand separation while positive supercoiling is introduced during DNA replication and transcription and must be corrected
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| Which enzymes accomplish DNA coiling in prokaryotes? | The topoisomerases: DNA gyrase (topoisomerase II in eukaryotes) and DNA topoisomerase I
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| What are the functions of supercoiling DNA? | 1. Compacts DNA 2.Negative promotes strand separation 3. Positive promotes DNA replication and transciption
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| Where is positive supercoiling introduced (with respect to the proteins) during DNA replication or transcription? | ahead of the protein doing the supercoiling
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| Where is negative supercoiling introduced (with respect to the proteins) during DNA replication or transcription? | behind the protein doing the supercoiling
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| Which enzymes correct for supercoiling and how? | Topoisomerase I: forms a single covalent bond with the DNA and breaks phosphodiester bond to allow free rotation
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| How does DNA gyrase correct the case of two double helices crossing over each other? | 1. makes a covalent linkage to both strands of one helix and making a double stranded break.
2. 2nd DNA is passed through break
3. break is resealed
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| What is chromatin? | complex of DNA and chromosomal proteins (histones)
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| What is a nucleosome core composed of? | H2A, H2B, H3, H4
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| A nucleosome is connected together by linker DNA and what protein? | H1 histone
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| How does the 30nm fiber form? | Interactions between adjacent histone H1 proteins.
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| Which base pair is methylated during transcriptional repression? | cytosine
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| List the antibiotic drugs that interfere with DNA gyrase | The quinalone family of antibiotics, including naladixic acid and ciprofloxacin, work by inhibiting DNA gyrase. A halt in DNA replication results due to positive supercoiling ahead of the replication fork.
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| Describe the structure of chromatin and histone proteins | Histones are rich in lysine and arginine, and as a result carry a positive charge. This makes it easier for negatively charged chromatin to associate.
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| What is the name of the prokaryotic version of histone protein? | HU protein
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| How do DNA and histones interact with eachother? | DNA is negatively charged and histones are positively charged and are therefore attracted to eachother.
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| Define euchromatin. | decondensed and transcriptionally active DNA
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| Define heterochromatin. | condensed and transcriptionally inactive DNA
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| What is the function of DNA methyltransferase enzyme | Methylation of cytosine to 5-methyl-cytosine
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| Describe the mechanism of DNA transcription repression. | Following DNA methylation, methyl cytosine binding proteins bind to the DNA, HDAC binds and removes acetyl groups off of histones giving them a greater positive charge and inducing supercoiling.
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| What happens to DNA when histones are acetylated. | euchromatic - active.
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| What components are required for synthesis of DNA? | 1. All four dNTPs 2. A fragment of DNA template 3. DNA poly 4. Mg2+ 5. primer with 3' OH
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| In what direction does DNA poly read DNA? | 3' to 5'
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| What nucleotide is acyclovir an anologue of? | guanine
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| Which enzyme converts acyclovir to acyclovir triphosphate and in what organism is it found in? | enzyme-thymidine kinase; organism-virus
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| Explain a tautomeric shift. | An unstable shift of cytosine to mimic a thymine. It then gets base paired with A.
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| Explain the significance of origins of replication. | site at which DNA replication occurs in both directions
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| What is leading strand synthesis? | Continual synthesis of DNA. Synthesis proceeds towards replication fork.
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| What is lagging strand synthesis? | DNA primase has to drop down a primer. Synthesis proceeds away from the fork.
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| What is the function of SSB proteins? | prevent re-annealing of DNA and prevent hairpin loops from forming
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| How are Okazaki fragments joined together? | DNA ligase which uses ATP.
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| What is the function of the Clamp protein? | tightly holds the DNA poly onto the template for synthesis of long templates and releases DNA poly when it stalls at a region of dsDNA
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| What drug inhibits bacterial DNA gyrase? | Ciprofloxacin
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| What is the function of telomerase and what kind of activity does it have? | It has reverse transcriptase activity. It has an RNA primer and binds it to the DNA overhang to extend. A primer can then be added so DNA poly can fill in that gap
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| What are the different types of RNA? | mRNA, tRNA, rRNA, snRNA
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| What is snRNA? | combines with certain proteins and is involved in RNA processing and gene regulation in eukaryotes
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| Describe the major steps of RNA synthesis in prokaryotes | 1. RNA polymerase binds -35 & -10 region, DNA molecule is unwound, sigma factor disassociates from core enzyme after 8-9 ribonucleotides have been polymerized 2.RNA polymerase unwinds DNA 3.transcription terminated at termination sequence
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| List the RNAs that are transcribed by the different types of RNA polymerases | RNA polymerase I (5.8S, 18 S, 28S rRNA genes), RNA polymerase II (all protein-coding genes like mRNA and some snRNA), RNA polymerase III (tRNA genes and some snRNA)
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| Explain how Courmarins and quinolones targets and inhibits specfic components of transcription. | inhibit bacterial gyrase, causing positive supercoils and subsequent halt of DNA replication and transcription
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| Explain how Rifamycin targets and inhibits specfic components of transcription. | – antibiotic from strain of streptomyces that inhibits transcription initiation by blocking formation of 1st phosphodiester bond, RNA pol of eukaryotes not affected
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| Is a nucleoside-monophosphate a nucleotide or nucleoside? | Nucleoside
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| How many charges does a nucleotide mono phosphate have? | 2 negative charges
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| What nucleoside triphosphate is involved in protein biosynthesis? | GTP
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| What nucleoside triphosphate is involved in lipid biosynthesis? | CTP
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| What nucleoside triphosphate is involved in carbohydrate biosynthesis? | UTP
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| What nucleoside triphosphate is involved in signal transduction? | GTP
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| What makes A-type DNA different from B-type? | bases are not perpendicular to axis, larger major groove and shallower minor groove.
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| When is A-type DNA observed? | When DNA solution is dehydrates, normally with short DNA, dsRNA and DNA/RNA hybrids
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| How do you denature DNA? | high temp or alkali conditions
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| What is the melting temperature (Tm)? | temp at which half DNA is denatured
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| What does azidothymidine and didanosine do? | block reverse transcriptase/do not have a 3' OH
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| What does acyclovir and tenofovir do? | They have an open chain sugar, acyclovir has a high affinity for viral DNA poly (herpes) and tenofovir has a high affinity for reverse transcriptase
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| What do the arabinosides do? | have a different planar arrangement and block DNA replication by DNA poly
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| What do the cytidine analogs do? | block DNA methylation
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| What are the differences between Prokaryotic and Eukaryotic mRNA? | bacterial mRNA are polycistronic and unmodified at 5' & 3' end
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| When are the RNA processing proteins transferred to the mRNA? | Processing proteins travel with RNA poly II and are transferred after initiation of transcription
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| What enzymes are involved in capping of mRNA? | phosphatase, guanylyl transferase, guanine-7-methyl transferase, 2'-O-methyl transferase.
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| What is the function of phosphatase? | removes phosphate from 5' end of RNA
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| What is the function of guanylyl transferase? | Adds GMP in reverse linkage
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| What is the function of guanine-7-methyl transferase? | Adds a methyl group to the 2'-O position to the secondlast base on the 5'end
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| What is the function of the methyl guanine cap? | helps cell distinguish between different RNA, plays a role in regulation of mRNA processing, transport, and translation
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| What proteins are involved in the modifying the 3' tail of mRNA? | CPSF, CstF and cleavage factors, PAP, PABP
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| What is the function of CPSF? | Binds to polyadenylation signal (AAUAAA)
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| What is the function of CstF? | binds GU-rich element
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| What is the function of cleavage factors? | bind (CA sequence) to cleavage site
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| What is the function of PAP? | Adds 200A to 3' end
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| What is the function of PABP? | binds to tail and assists in directing translation
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| What are the steps of intron removal? | 1. U1 binds 3'GU 2. U2 binds Branch point A 3. U4/6 and U5 bind U1 and U2 3. U4 released 4. 3'GU cleaved and G binds A 5. 3' AG cleaved, Exons ligated, spliceosome disassembles
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| What causes Systemic Lupus Erythematosis? | antibodies to U1 snRNA
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| What causes beta-thalasemmia? | mutations in the beta-globin gene that generate additional splice sites within the mRNA
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| What causes Limb Girdle Muscular Dystrophy? | Mutation in calpain 3 gene that generates a new splice site within the exon 16
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| What can control alternative RNA splicing? | weak splice site sequences, negative/positive control
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| What does the cytidine deaminase enzyme do? | introduces a premature stop codon by alternative splicing into APO-B in intestine to make shorter protein.
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| What does adenosine deaminase do? | Changes A to I in glutamate receptor to change glutamine to arginine in brain
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| How is protein written by RNA poly and DNA poly? | 5' to 3', N terminus to C terminus
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| Which RNA consitutes for 99.9% of total RNA? | mRNA, tRNA & rRNA
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| What does redundant genetic code mean? | several codons code for one amino acid
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| What does non-overlapping genetic code mean? | each codon is read three at a time without overlap
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| What is an inframe mutation? | When entire codons are removed or inserted
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| What are the three stop codons? | UGA, UAG, UAA
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| What is the start codon? | AUG
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| What is the wobble hypothesis? | predicts the base pairing of the 5' anticodon and 3' codon.
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| Which protein does the anticode IGC carry? | alanine
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| Is inosine a purine or a pyrimadine? | purine
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| what is a a charged tRNA? | a tRNA with its amino acid group
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| What enzyme calatyzes the activation of an amino acid? | aminoacyl tRNA synthetase
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| What drives the formation of a charged tRNA? | hydrolysis of pyrophosphate
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| What are the 5 steps of formation of a polypeptide polymer from an RNA template? | 1. Activation of the monomer 2. Initiation 3. Elongation 4. Termination 5. Processing the polymer
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| In prokaryotes, after methionine is activated how what molecule donates it's carbon to make formylmethionine? | N-formyltetrahydrofolate
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| In prokaryotes, how is the correct start codon recognised? | 30S binds to the shine delgarno sequence
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| What catalyzes the translocation of the mRNA relative to the ribosome? | EF-G or EF-2 with GTP
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| How is the terminal amino acid removed from the ribosome? | Release factor allows peptidyl transferase to cleave ester bond between tRNA and amino acid
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| What are the three differences between prokaryotic and eukaryotic translation? | prokaryotic:polycistronic, translation and transcription together, internal AUG sites used
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| How is protein synthesis affected by streptomycin? | Binds to 30S/prevents assemble of ribosome
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| How is protein synthesis affected by tetracycline? | Binds to 70S/prevents access to incoming aminoacyl-tRNA
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| How is protein synthesis affected by erythromycin? | Binds to 70S/Block elongation
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| How is protein synthesis affected by chloramphenicol? | Inhibits peptidyl transferase/blocks elongation
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| How is protein synthesis affected by cyclohexamide? | blocks elongation in eukaryotes
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| How is protein synthesis affected by puromycin? | Blocks elongation after forming a peptide bond chain causing peptide to fall out of ribosome P-site
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| How is trypsinogen cleaved to trypsin? | enteropeptidase
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| What receptor is a tyrosine kinase? | the insulin receptor
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