Replication/Transcription/Translation Enzymes and More

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In E. coli, which DNA Polymerase has 5' to 3' exonuclease activity? Why is this important?: DNA Pol I; important because Pol I is responsible for degrading the RNA primer s of the Okazaki fragments and replacing them with deoxyribonucleotides.
What direction does proofreading proceed?: 3' to 5' becaues the excision of an incorrectly paired base must be done in the reverse direction
What is the difference between 5' to 3' exonuclease activity and 3' o 5' exonuclease activity?: 5' to 3' can removie either ribonucleotides or deoxyribonucleotides and can remove more than one at a time while 3' to 5' removes only incorrectly matched deoxyribonucleotide base pairs from both the leading and lagging strand.
DNA Ligase: Seals nicks in DNA between a 5' phosphate and a 3' OH
What enzyme fills the gaps between the end of one Okazaki fragment and the RNA primer of the "next" fragment? Was the "next" fragment synthesized before or after?: DNA Polymerase I; the "next" fragment was synthesized first.
In E. coli, what enzyme is responsible for DNA chain elongation? In eukaryotes?: DNA Pol I; DNA Pol δ
What eukaryotic enzyme is responsible for primase activity and initiatin of DNA synthesis?: DNA Pol α -- it synthesizes a short RNA primer that is extended by its elongation activity. Pol δ is then recruited.
What do Pol beta, epsilon, and gamma do?: Beta and epsilon are responsible for DNA repair. Gamma replicates mitochondrial DNA.
How does telomerase preserve the ends of chromosomes?: 1. Telomerase extends the 3' end of the DNA
2. Telomerase contains an RNA template and primase activity, which synthesizes a RNA primer complimentary to the 3' overhang of the chromosome
3. The 3' end of the primer serves as a primer for DNA Polymerase, which fills the gap between the primer and the 5' end of the chromosome
4. RNA primer is removed (thus, 3' end is still longer than 5' end by the length of the RNA primer)
What enzyme removes RNA primers from new DNA in eukaryotic cells?: RNase H
Pyrimidine dimers are removed via what mechanism?: excision repair: UV-specific endonuclease recognizes the dimer, cleaves the damaged stran on both sides of the dimer, releases the damaged oligonucleotide. The gap is filled is filled by a 5' to 3' DNA Polymerase and sealed by DNA ligase.
In base excision repair, abnormal bases are removed by glycosylases. What remains after this cleavage.: Glycosylases removes the nitrogenous base from the sugar-phosphate backbone, leaving a apyrimidic/apurinc site (AP-site)
AP endonuclease does what?: recognizes that a base is missing and makes a cut to the 5' side of the AP site. Then a deoxyribose-phosphate lyase removes the empty sugar-phosphate residue. DNA Pol and DNA ligase complete the repair process.
What differentiates a new strand of DNA from an old strand?: The parental strand is methylated but the new strand is not (temporarily)
Difference between Topoisomerase I and II: Topo I cuts one strand of DNA in helix while Topo II cutes both strands. Both allow strands to pass through one another and allows the release of twisting tension.
The RNA transcript is identical to?: non-template strand
Do prokaryotes have just one or multiple RNA Pols?: one: has 5 subunits. Sigma subunit recognizes the promoter and dissociates from the rest of the enzyme shortly after transcription begins
Mechanism of RNA Pol?: Recognizes and bind promoter, then opens the DNA dbl helix, begins synthesizing RNA and continues until a termination signal is reaches
Formation of a hairpin structures followed by a string of U's in transcription termination is rho dependend or independent?: independent
What is rho dependent termination?: A rho protein recognizes a termination signal in the new RNA, thus releasing RNA Pol and its transcript.
RNA is synthesized in what direction?: 5' to 3'
Are specific termination sequences always used in eukaryotes?: No, the polymerase can continue for several hundred bases. RNAse generates the correct 3' end of the transcript.
How many RNA Pols are in eukaryotes? Where are they located and what are their products?: 3; RNA Pol I is in the nucleolus and synthesizes rRNA; RNA Pol II is in the nucleus and synthesizes hnRNA; RNA Pol III is in the nucleus and synthesizes tRNA, 5S rRNA, and snRNA.
What sequence on tRNA binds amino acids and where is it located? Is the amino acid attached through its carboxyl or amino terminus.: C-C-A; located on the 3' end of tRNA; attached through carboxyl group
Where does capping occur?: 20-30 nucleotides after the beginning of the hnRNA transcript
polyadenylation: addition of a long tail of AMP residues to the 3' end of hnRNA...ONLY in eukaryotic cells
Is the Poly-A tail encoded by the genome? What is its purpose?: No, there is not a template for the tail. It is thought to be involved in stability and transport.
What is composed of RNA-protein complexes termed small nuclear ribonucleotproteins (snRNPs)?: spliceosomes (composed of snRNPs U1, U2, U4, U5, U6)
5' and 3' dinucleotides of intron?: exon1-5'GU-----AG 3'-exon2
U1 and U2 bind where?: U1 binds to a sequencee at the 5' splice site while U2 is paired near an A nucleotide at a branch point of the intron
What is the function of the spliceosome?: brings the slice sites into close proximity and forms a large RNA loop (lariat)
What is the bond that makes the lariat structure?: 2'-5' bond between the 5' end of the intron and the A nucleotide in the branch region
What happens after the lariat is formed?: The 3' end of the intron is cleaved after the AG sequence, releasing the lariat. Then the free 3' and 5' ends of the exon are joined.
What is attenuation?: a process in prokaryotic cells in which transcription is prematurely terminated by translation
enhancer: a DNA sequence that affects the rate of transcription through beind to regulatory proteins
operator: a DNA sequence in bacterial operons to which regulatory proteins bind
operon: promoter + operator + structural genes
polycistronic RNA: single RNA containing multiple genes; only in prokaryotes
repressor: protein that binds to an operator and prevents transcription of the associated gene
TATA box: DNA sequence in eukaryotes that is bound by transcription factors
transcription factor: DNA binding proteins that are required for transcription or that increase the rate of transcription
When is Lac repressor produced?: ALWAYS
What is necessary if the lac genes are to be expressed?: Lac I (repressor) must be bound by the inducer allolactose.
Will lac genes be transcribed if glucose is present?: No, operon is repressed in presence of glucose because cAMP production is inhibited.
What is involved in induction of the lac operon?: cAMP is produced when lactose is present. cAMP bind to the cAMP receptor protein (CAP). CAP-cAMP complex binds alongside the RNA Pol binding site of the lac operator and facilitates binding of RNA Pol.
What two factors are absolutely necessary for lac gene expression?: cAMP and allolactose (inducer)
High levels of Trp act as what?: co-repressor of the Trp operon
trp + trp repressor: binds to operator of trp operon to inhibit production of trp
In the trp operon, what is the leader sequence?: sequence upstream to the first trp gene and it requires trp for synthesis.
what happens if trp is absent from the cell?: since the leader peptide sequence requires trp, the ribosome stalls until a charge trp-tRNA binds to the ribosome. Stalling produces a hairpin loop between domains 2 and 3, prohibiting formation between domains 3 and 4 and thus probhibiting termination of transcription.
What is the importance of ribosome stalling?: If trp is absent or at low levels, more trp is needed. If the ribosome didn't stall, transcription would be terminated and trp would never be produced.
How do enhancer bind proteins activate transcription?: by physically touching the basal transcription complex located at the promoter/TATA box or by altering the chromatin structure near the promoter site
Promoter: region which must be recognized and bound by RNA Pol to allow transcription
CCAAT box: regulates the efficiency of promoter usage and therefor the frequency of transcription of the gene; bound by CTF; located at -80
What is TFIID and what does it bind?: complex of transcription factors; bind TATA box (located at -30)
What part of TFIID binds the TATA box?: TATA box binding protein (TBP); also associated with TAFs (TATA-associated factors)
TBP andTAFs promote transcription by doing what?: recruiting additional transcription factors
ATG: sequence that will be AUG (start codon) in mRNA and encode the first amino acid of the protein
length of mRNA between the CAP site and the AUG: 5' untranslated region
TAA, TAG, TGA: sequences that become termination signals in mRNA
AATAA: polyadenylation signal
length of mRNA between the termination signal and the poly-A tail: 3' untranslated region
Zinc finger: contains zinc bound to Cys and His side chains; receptors for steroid hormones
a compound that can be secreted by one cells and transmit a signal to another cell: inducer
IkB-NFkB complex is dissociated when?: B cell is activated
What effect does IkB have on NFkB? What is NFkB's function in the B cell?: IkB inactivates NFkB which interacts with the Ig light chain promoter to activate gene transcription.
mechanism of steroid hormones: Lipid soluble hormones enter cell and bind to receptors, binding releases receptors from inhibitors and the receptors dimerize then translocate to the nucleus and activate transcription.
Amino acyl tRNA synthetase: charges tRNA with the correct amino acid
What are the two binding sites of amino acyl-tRNA?: One for tRNA, one for ATP
What type of bond is formed between the amino acid and the 3' hydroxyl group of the tRNA?: aminoacyl ester linkage
How many amino acyl-tRNA synthetases are there for one amino acid: 1
Can an amino acid have more than one tRNA specific for it?: Yes- DEGENERACY
Can a codon code for more than one AA?: No- UNAMBIGUOUS
What is the thymidine-psuedouridine-cytidine loop involved in?: ribosome binding
What is the dihydrouracil lopps important for?: recognition of a given tRNA species by its correct amino acyl-tRNA synthetase
Accoriding to wobble, what position on what molecule can be variable?: the 5' position of the anitcodon (amino acids that have more than one codon are most often different at the 3' position)
Peptidyl transferase: enzymatic activity of the 60S subunit that forms the peptide bond
components of prokaryotic ribosome: 30S + 50S = 70S
components of eukaryotic ribosome: 40S + 60S = 80S
What doe sthe 30S subunit bind to?: initiation factors 1 and 3
What is the importance of 30S binding to IF-1 and IF-3?: ensures that the small ribosomal subunit will not bind with the alrge 50S subunit until it has properly bound the mRNA molecules
What does IF-2 do?: binds to the 30S subunit and recruits the initiator tRNA molecule
What is the difference between prokaryotic and eukaryotic initiator tRNA?: prokaryotic initiator tRNA is converted to a N-formylmethionine molecule
Shine-Dalgarno sequence: ribosome binding site that that 30S subunit, 3 IFs, and tRNA bind to to set reading frame
What happens after binding of the Shine-Dalgarno sequence?: tRNA basepairs with the AUG start codon and IF-3 is released.
30S preinitiation complex: 30S + IF-1 + IF-2 +tRNA
When can the 50S subnit bind to the preinitiation complex?: when IF-3 is released
IF 1 and 2 are released when?: when the 70S ribosome is formed
A-site: where the incoming aminoacyl-tRNA enters the ribosome
1st step of chain elongation: correct aminoacyl-tRNa is positioned in the A site
2nd step of peptide chain elongation: transpeptidation
3rd step of peptide chain elongation: translocation- shift along mRNA by one codon relative to ribosome
What is the attachement point for the growing peptide chain?: peptidyl tRNA--the tRNA in the P site
EF-Tu is required for?: binding of second aminoacyl-tRNA to A site
where does the energy for peptide bond formation come from?: energy rich aminoacyl-tRNA bond
Direction of transfer in formation of peptide bond?: Peptide chain in P site is transfered TO the A site. Peptide is now located in A site and translocation moves it to the P site. A site is then empty.
Why does termination occur when a nonsense codon enters the A site?: there are no charged tRNA molecules that recognize stop codons
What enters the A site when there is a nonsense codon?: releasing factors
What two RFs recognize different stop codons?: RF 1 and RF 2
What does RF 3 do?: assists RF 1 and RF 2 with polypeptide release by binding GTP and forming a herterodimer with RF 1 or RF 2
Alteration of peptidyl transferase activity results in what?: hydrolyzation of the bond linking the peptidyl-tRNA and the nascent polypeptide, releasing the new polypeptide
Do eukaryotes use the Shine-Dalgarno sequence?: No, the ribosome scans the mRNa unitl it finds the appropriate start codon
preferred start site for eukaryotic translation: Kozak consensus sequence
How many releasing factors are there in eukaryotes?: 1
What regulates inititiation factors in eukaryotes?: phosphorylation; e.g.: eIF2 is inactivated by phosphorylation by PKR when amino acid pool is deprived

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