Biol 280
What does 16S rRNA in the small subunit do? What is it part of?
Contains a sequence complimentary to the Shine-Delgarno sequence in the mRNA, helps line up the mRNA and AUG (methionine) codon on the ribosome. Part of the 30S subunit.
What are four DNA binding motifs found in DNA binding proteins?
Helix-turn-helix; zinc finger; homeodomain; leucine zipper; basic helix-loop-helix
Heme is degraded by what enzyme?
Heme oxygenase
What type of damage to DNA would base excision repair be used to fix?
Damaged bases (i.e., methylation)
What type of mutation is a reason to keep uracil in RNA versus thymine?
Deamination
What is the DNA Polymerase III complex responsible for lagging strand synthesis composed of?
Dimeric Beta-clamp, 3 ATP and hydrolysis allows Beta-clamp to close around DNA.
What are the mechanisms by which RNA can be decayed
Endonuclease cleavage or deadenylation-independent decapping (nonsense-mediated decay)
True or false? RNA polymerase requires a primer to initiate synthesis at promoters.
False
True or false? Short period after replication, newly synthesized strand has methylation marks but parental strand does not.
False
True or false? Transcription does not involve promoters in eukaryotic gene expression regulation.
False
True or false?The σ (sigma) subunit of E. coli RNA polymerase is a proofreading exonuclease.
False
True or false? Correction of mismatch involves MutL-MutS complex which cleaves the unmodified strand.
False, MutH cleaves
True or false? Enhancers are DNA binding proteins.
False. Enhancers are DNA sequences that bind to activators
True or false? Operators are DNA binding proteins.
False. Operators are DNA sequences that bind repressors.
True or false? Polycistronic transcripts are common in both bacteria (prokaryotes) and eukaryotes.
False. Polycistronic messages are common in prokaryotes only; eukaryotes have monocistronic messages
How do cells utilize iron?
Fe•S proteins, Heme synthesis, and other Fe-proteins.
What is DNA Pol I?
Fills in gaps where RNA existed. Contains a 5'→3' exonuclease that removes RNA primers
What are two ways DNA structure is stabilized?
First - H-bonding between bases and Second - Stacking interactions between bases (since there are flat and aromatic).
What are the two experiments that prove that DNA stores genetic information?
First Experiment - DNA purified from the heat-killed virulent bacteria, when mixed with live non-virulent strain, resulted in a strain of live virulent strain that remained virulent in subsequent generations. Second Experiment - Radioactively labeled DNA (32P) and coat protein (35S) and found that radioactive progeny virus with genetic material was 32P
How are prokaryotic and eukaryotic translational initiation similar and different.
For both, soluble factors regulate initiation, Eukaryotic mRNAs are monocistronic and lack a ribosome binding site
Is DNA polymerization reversible?
Hydrolysis of pyrophosphate makes it essentially irreversible
What are the steps in protein synthesis requiring GTP hydrolysis?
Initiation: IF2-GTP → IF2-GDP Elongation (proofreading): EF-Tu-GTP → EF-Tu-GDP Elongation (translocation): EF-G-GTP → EF-G-GDP Termination: EF-G-GTP → EF-G-GDP
Why is the genetic code degenerate?
More than one codon codes for an amino acid, because (4³) = 64 and we only have 20 a.a.
Can RNA polymerases proofread?
No 3' → 5' exonuclease activity so no proofreading
The lagging strand synthesis is Discontinuous. Each fragment of RNA primer and DNA is called what?
Okazaki fragment
What type of damage to DNA would direct repair be used to fix?
On-site damage repair (i.e. Single methylation)
What are the steps of mismatch repair?
Parental strand marked (methyl) to distinguish old and new; mismatched bp forms a kink. Unmethylated strand nicked. DNA degraded from Me past mismatch. Pol III fills in DNA.
What is the role of peptidyl transferase in bacterial protein synthesis?
Peptidyl transferase catalyzes peptide bond formation during each elongation cycle. (Catalysis is thought to be RNA-based and rely on 23S rRNA.)
What is the role of peptidyl transferase in bacterial protein synthesis?
Peptidyl transferase is a ribozyme in the 50S ribosomal subunit. It catalyzes formation of each peptide bond as the ribosome moves along the mRNA.
Does a single RNA polymerase transcribe genes that encode mRNAs, tRNAs, and rRNA in eukaryotes, prokaryotes, or both?
Prokaryotes
Does the sigma (σ) subunit detach from RNA polymerase after transcription has initiated in eukaryotes, prokaryotes, or both?
Prokaryotes
How do prokaryotic and eukaryotic mRNA expression compare with regards to organization of genes into operons?
Prokaryotic genes are often arranged into operons, while eukaryotic genes are not.
In eukaryotes what is the purpose of a telomere and how are they added?
Stabilize the end of linear chromosomes, by telomerase
Where does RNA Pol bind to initiate transcription?
The promoter
What provides barriers against RNA Decay?
5' caps and poly(A) tails
What bond connects the mRNA to 5' cap?
5'-5' triphosphate linkage
After binding by E. coli RNA polymerase, what is the correct order of events for transcription initiation?
Closed complex formation, open complex formation, start of RNA synthesis, promoter clearance.
How do prokaryotic and eukaryotic mRNA expression compare with regards to coupling between transcription and translation?
Co-transcriptional translation occurs in prokaryotes, but not in eukaryotes due to compartmentalization.
What are exons?
Coding (Expressed) mRNA sequences that are spliced together.
Which nucleotide(s) regulate the conversion of Ribose-5-phosphate to PRPP?
ADP
Which nucleotide(s) regulate the conversion of IMP to adenylosuccinate?
AMP
In base excision repair, what removes the sugar?
AP endonuclease
What is the role of ATP in bacterial protein synthesis?
ATP is the substrate for aminoacyl-tRNA synthetases; it donates an AMP residue in the formation of aminoacyl adenylate, which donates the aminoacyl group to tRNA.
Which nucleotide(s) regulate the conversion of CDP to dCDP?
ATP, dTTP
Which nucleotide(s) regulate the conversion of GDP to dGDP?
ATP, dTTP
What are the purine nucleobases?
Adenine and guanine
Why use alternative RNA processing?
Allows organism to make a diversity of protein products from one gene. Can produce Multiple mRNAs from a single primary transcript.
What stage(s) of protein synthesis do aminoacyl-tRNA synthetases interact with?
Amino acid activation
What is the role of aminoacyl-tRNA synthetases in bacterial protein synthesis?
Aminoacyl-tRNA synthetases both activate an amino acid for protein synthesis and pair an amino acid with its appropriate tRNA.
How is aminolevulinate turned into tetrapyrroles?
Aminolevulinate is dimerized, then the product is tetramerized, to form tetrapyrroles
List one basic property that distinguishes RNA polymerases from DNA polymerases, and list one basic property they share.
Among the distinguishing characteristics: RNA polymerase does not require a primer, but DNA polymerase does; RNA polymerase lacks the 3' ® 5' proofreading exonuclease activity present in DNA polymerase. Among the shared properties: both enzymes use nucleoside triphosphates as substrates, require Mg 2+ and Zn2+, produce an antiparallel complement to the template, and synthesize nucleic acids in the direction 5' ® 3'.
16s RNA
component of small 30s subunit - contains sequence complementary to Shine Dalgarno sequence in mRNA, helps line up mRNA imitation AUG codon on ribosome
EF-TU
elongation factor, catalyzes binding of AA-tRNA to ribosome (elongation phase)
activator
enhance access of RNA pol to DNA
ribonucleases
enzymes made of RNA that break down RNA molecules
How are genes arranged in prokaryotes?
in operons
Puromycin is an antibiotic that inhibits protein synthesis because why?
it looks like aminoacyl-tRNA and binds to the A site.
What type(s) of RNA does Pol II transcribe?
mRNA, snRNA, miRNA
cDNA library
made as DNA copy of mRNA - contains only DNA sequences that are expressed in cell
promoter
regulatory DNA sequence where RNA pol binds
operator
regulatory DNA sequence where repressor can attach and keep RNA pol from being able to perform transcription
Give an example of a transition and transversion base substitution.
Transition is Purine to Purine or Pyrimidine to Pyrimidine. Transversion is Pyrimidine to Purine or vice versa. Purines are A and G ; Pyrimidines are T and C.
If DNA strands break (nick) then what is the ΔLK?
Undefined. LK specifies the number of helical turns in a closed circular DNA
Which is typically the "wobble" position?
Usually the 3rd base pair (5' position of the anticodon, 3' position of codon"
Define "constitutive", with respect to regulation of transcription.
constant or unregulated transcription
genomic library
contains all sequences present in chromosomes- including DNA sequences not transcribed
Is leading strand replication continuous, discontinuous, or semidiscontinuous?
continuous (and in the direction of the movement of the fork)
biological amines
derived from decarboxylation of Das
Howard Dintis expt
add radiolabeled Ile-tRNA to translating ribosomes- at low time intervals: high concentration at C terminal, at higher time intervals, more at N terminal, shows that peptide synthesis is initiated at N terminal, terminated at C terminal
thymidylate
adds methyl group
purine bases
adenine, guanine
direct repair
alkylated bases can be directly repaired, transfer methyl onto enzyme
if the anticodon of the tRNA is (5') UGG (3') what codons will be recognized?
3'-ACC-5' AND 3'-GCC'5'
Approximately how many high-energy phosphate bonds must be consumed to incorporate one amino acid into a protein (i.e., per peptide bond)?
4
What sequences regulate translation of ferritin mRNA?
5' UTR sequence, reporter constructs, CAT: Chloramphenicol Acetyl Transferase
What is the essential difference between a genomic library and a cDNA library?
A genomic library contains (in principle) all of the sequences present in the chromosome(s), including DNA sequences that are not transcribed. Because a cDNA library is made as a DNA copy of mRNA, it contains only those DNA sequences that are expressed in the cell.
In base-excision repair, what is the first enzyme to act?
DNA glycosylase
What are the steps of base excision repair?
DNA glycosylase cleaves off base. AP endonuclease removes sugar. Pol I fills in gap. Ligase seals the nick.
What is the first enzyme to act in base-excision repair?
DNA glycosylase.
The nicks left behind after DNA synthesis are sealed by what enzyme?
DNA ligase
What seals the nicks formed in DNA replication?
DNA ligase (Uses ATP!)
What removes the RNA primers AND replaces the with DNA?
DNA pol I exonuclease activity 5' to 3'
What is the name of the enzyme that removes the RNA primer in DNA synthesis?
DNA polymerase I
What is the origin?
The point of initiation of synthesis
Which topoisomerase changes LK in increments of 1?
Type 1
Which topoisomerase cleaves one strand of duplex DNA?
Type 1
Which topoisomerase removes negative supercoils?
Type 1
Which topoisomerase change LK in increments of 2?
Type 2
Which topoisomerase cleaves both strands of DNA?
Type 2
Which topoisomerase hydrolyzes ATP?
Type 2
Which topoisomerase introduces negative supercoils?
Type 2
What type of mutation generates thymine dimmers?
UV irratiation
Briefly describe DNA damage induced by UV radiation. Mention what happens chemically, and which kind of repair mechanism is used to repair this kind of damage.
UV radiation induces the formation of pyrimidine dimers (cyclobutene thymine dimers). These dimers cause large distortions in the DNA and are repaired by nucleotide-excision repair.
How do cells uptake iron?
a TfR protein binds to four Fe atoms.
The degradation of heme by heme oxygenase yields what?
biliverdin
What two substances is heme broken down into?
biliverdin and bilirubin
What kind of reaction happens in Group II intron splicing?
transesterification
How do cells regulate iron?
"labile iron pool"!
How many polymerases do eukaryotes have?
3
What are the steps of prokaryotic translation initiation?
1) 30S binds IF-1 and IF-3. 2) mRNA binds-positioned by 6S rRNA. 3) IF-2•GTP binds, fMet-tRNA^fMet binds. 4) 50S binds, IF-2•GTP hydrolyzed, IF-1, IF-2, IF-3 released.
what are the steps for naming ribonucleotides?
1. Check for oxy groups (d or not) 2. check what base (A G C U) 3. check # phosphates (M, D, T)
What is the role of 16S RNA in bacterial protein synthesis?
16S RNA contains a sequence complementary to the Shine-Dalgarno sequence in the mRNA, and helps to line up the mRNA initiation AUG codon on the ribosome.
What is the role of 16S RNA in bacterial protein synthesis?
16S RNA is a component of the small (30S) subunit. It contains a sequence complementary to the Shine-Dalgarno sequence in the mRNA, and helps to line up the mRNA initiation AUG codon on the ribosome.
Briefly describe the Wobble hypothesis
1st two bases of an mRNA codon always form strong base pairs. The 5' base of the anticodon determines the number of codons. A 5' base C can pair with G. A 5' base A can pair with U. A 5' base U can pair with A or G. A 5' base G can pair with C or U. A 5' I base can pair with A, U, or C.
What is the linking number of a relaxed closed-circular DNA molecule with 5,000 base pairs? Assume that the DNA is a right-handed helix with 10 base pairs per turn, and that one round of reaction equals one turnover.
500
The difference between a ribonucleotide and a deoxyribonucleotide is what?
A deoxyribonucleotide has an —H instead of an —OH at C-2.
What is an Okazaki fragment?
A segment of DNA that is an intermediate in the synthesis of the lagging strand.
What is the sequence of events that occurs during the synthesis of the lagging strand in replication?
Action of primase to synthesize primer, synthesis of lagging strand by DNA polymerase III, DNA polymerase I action to remove primers, linkage of Okazaki fragments by DNA ligase.
What are the 5 stages of protein synthesis?
Activation, Initiation, Elongation, Termination, Folding/posttranslational modification
What kind of mutation might be fixed by direct repair?
Alklylation (like O⁶- methylguanine)
Where does the hydroxy group come from for Group I splicing?
An external hydroxyl-guanosine (attacks the 5' end)
How is an origin of replication used in cloning of a plasmid?
An origin of replication assures that the plasmid will replicate autonomously in the bacterium.
How are antibiotic resistance genes used in cloning of a plasmid?
Antibiotic resistance allows a researcher to select for a bacterial cell clone that carries the plasmid; loss of an antibiotic marker in a strain known to contain the plasmid can be used to infer the presence of a cloned DNA segment that interrupts the antibiotic resistance gene.
where is mature mRNA polyadenylated?
At the 3' end
where is mature mRNA capped?
At the 5' end
Briefly explain the differences between base-excision repair and nucleotide-excision repair.
Base excision repair involves removing only the defective base from the DNA by cleavage of the N-glycosidic linkage of the base to the deoxyribose. This leaves and apurinic or apyrimidinic ( AP or abasic) site. The sugar is then removed, leaving a nick in one strand. Nucleotide excision repair involves removing the defective base, along with neighboring nucleotides by cleavage of 2 phosphodiester bonds in the DNA chain, leaving a gap.
Which nucleotide(s) regulate the conversion of Aspartate to N-Carbamoylaspartate?
CTP
How is purine ring synthesis regulated?
CTP inhibits the committed step which is catalyzed by aspartate transcarbamoylase
Promoter sequences generally contain more A-T base pairs than G-C base pairs. Explain this.
Because A-T base pairs are stabilized by only 2 hydrogen bonds (compared with 3 for G-C base pairs), open complex formation by RNA polymerase (unwinding) is more favorable in double-stranded regions rich in A-T base pairs.
Does RNA polymerase initiate transcription de novo, with no requirement for a primer, in eukaryotes, prokaryotes, or both?
Both
What are the similarities between RNAPs and DNAPs?
Both require nucleoside triphosphates, Mg, Zn, synthesize 5' to 3', and produce a complement to the template.
The linking number (Lk) of a closed-circular, double-stranded DNA molecule is changed by what?
Breaking a strand, unwinding or rewinding the DNA, then rejoining it.
How are mRNA splice sites specified?
By sequences at the end of introns
What is the sequence for a PstI restriction site?
CTGCA|G G|ACGTC
What does DNA polymerase actually do?
Catalyzes the formation of the phosphodiester bond (nucleophilic attack of the 3' OH)
What is the effect of methylation on base pairing?
Changes base pairing properties (e.g., Guanine ≡ Cytosine after methylation and replication becomes O⁶-Methylguanine = Thymine).
What does ribonucleotides reductase do?
Converts ribonucleotides to deoxyribonucleotides
When a mismatch is introduced in a double stranded DNA during bacterial DNA replication, the methyl directed repair system does what?
Corrects the mismatch by changing the newly replicated strand.
In order to perform PCR, what reagents must be included?
DNA fragment, primers flanking the region of interest, dNTPs, DNA Polymerase.
PCR requires
DNA fragment, primers flanking region of interest, dNTPs, DNA polymerase
In base excision repair, what cleaves the N-glycosyl bond?
DNA glycosylase
DNA Replication is preformed by what?
DNA polymerases
template strand
DNA strand makes mRNA
What did the Meselson-Stahl experiment determine?
DNA synthesis in E. coli proceeds by a semiconservative mechanism.
Describe chromosome compaction.
DNA to beads on a string form to 30 nm fiber to one loop to loops attached to a nuclear scaffold eventually to coils and formation of two.
Briefly describe how DNA-binding proteins interact with DNA, where they typically bind in the double helix, and name at least two DNA-binding motifs.
DNA-binding proteins interact with DNA by forming hydrogen bonds between amino acid side chains and bases. They generally bind to DNA in the major groove. Helix-turn-helix; zinc finger; homeodomain; leucine zipper; basic helix-loop-helix
What type of mutation occurs via agents such as dimethylnitrosamine, dimethylsulfate?
Depurination
In 1961, Howard Dintzis carried out an experiment that defined the direction of polypeptide chain growth during protein synthesis in cells. The experiment involved the analysis of hemoglobin molecules that were being synthesized in reticulocytes in the presence of radioactive amino acids. Describe the experiment.
Dintzis added radiolabeled Ile-tRNA to actively translating ribosomes. In the experiment, only completed polypeptides were isolated and analyzed for the amount of radioactivity in different regions. The greatest radioactivity should be located in the region furthest from the initiation point and the least radioactivity in the region closest to the initiation point. The opposite is true for the termination point. The analysis showed that polypeptide synthesis was initiated at the amino terminus and terminated at the carboxyl terminus.
What happens in the initiation of DNA replication?
DnaA opens the duplex ori. DnaC loads DnaB (helicase) onto ori where it unwinds the DNA. DNA gyrase then relieves the strain through topoisomerase II activity.
What are the main features of direct repair?
Does not remove base or nucleotide. Repairs defect BUT inactivates one protein per repair (expensive).
How is epinephrine made?
Dopamine is hydroxylated to from norepinephrine which has a methyl group added to it to form epinephrine
What is the role of EF-Tu in bacterial protein synthesis?
During elongation, EF-Tu-GTP binds to the incoming aa-tRNA, which base-pairs with the anticodon in the A site. If the fit is good, GTP hydrolysis occurs (proofreading).
What is the role of elongation factor G (EF-G) in bacterial protein synthesis?
EF-G participates in the translocation of the ribosome down the mRNA by one codon after each peptide bond is formed using a conformational change catalyzed by GTP hydrolysis.
What type of restriction site is CTTAA|G a sequence for?
EcoRI
What type of restriction site is G|AATTC a sequence for?
EcoRI
What type of restriction site is CTA|TAG a sequence for?
EcoRV
What type of restriction site is GAT|ATC a sequence for?
EcoRV
What stage(s) of protein synthesis does EF-Tu interact with?
Elongation
What is the purpose of DNA Pol III's Beta clamp?
Enables the high processivity of DNA Pol III by keeping it bound longer.
Do promoter sequences have little intrinsic affinity for RNA polymerase in eukaryotes, prokaryotes, or both?
Eukaryotes
Is there is a cap consisting of a guanosine joined to the 5'-terminal nucleotide through a 5' to 5' triphosphate group at the 5' end of the mature mRNA in eukaryotes, prokaryotes, or both?
Eukaryotes
How do prokaryotic and eukaryotic mRNA expression compare with regards to processing of the primary transcript?
Eukaryotic mRNAs are processed after transcription, while prokaryotic mRNAs are not.
True or false? A lariat intermediate is formed during group I intron splicing.
False
True or false? All three eukaryotic RNA polymerases are involved in mRNA synthesis.
False
True or false? DNA in the promoter region must form an open complex before RNA polymerase can bind to it.
False
True or false? Eukaryotic cells contain at least five DNA polymerases all of which are responsible for nuclear DNA replication.
False
What are the steps of nucleotide excision repair?
Exinuclease makes 2 cuts to remove damaged DNA. Polymerase & ligase replace DNA.
What does 23S rRNA do?
Has peptidyl transferase aka. It catalyzes formation of peptide bond during each elongation cycle.
What is the sequence for a EcoRV restriction site?
GAT|ATC CTA|TAG
Which nucleotide(s) regulate the conversion of IMP to XMP?
GMP
Briefly describe why it is crucial to repair the alkylation product O⁶-methylguanine in DNA and indicate the repair mechanism to fix this type of DNA damage.
Guanine methylation changes its base pairing properties. mG pairs with T instead of C, which can introduce a mutation during DNA replication. O⁶-methylguanine is repaired by direct repair.
What do snoRNAs do?
Guide cleavage and nucleoside modification of rRNA precursors.
What is the sequence for an EcoRI restriction site?
G|AATTC CTTAA|G
Describe briefly the relationship between chromatin structure and transcription in eukaryotes.
I eukaryotic chromosomes promoter access is restricted. Condensed chromatin is inaccessible and must be remodeled. Remodeling can occur through covalent modification of histone proteins and by enzymes that actively displace nucleosomes.
What is the role of initiation factor 2 (IF-2) in bacterial protein synthesis?
IF-2 is a protein factor that, when bound to GTP, brings the fMet-tRNAfMet to the initiation complex.
If only lactose is in the growth medium, what proteins will be bound to the lac operon regulatory region?
If only lactose is present, CRP will be bound.
What are the three major stages of DNA replication?
Initiation, elongation, termination
What are the functions of biological amines?
Important functions as hormones and/or neurotransmitters
Compare and contrast ρ-dependent and ρ-independent termination of transcription in prokaryotes.
In both, the transcription complex pauses at specific sequences in the DNA template. In ρ-independent termination, the formation of hairpin structures in addition to formation of A-U base pairing causes pausing which leads to dissociation of the product RNA and the polymerase from the template DNA and hence to termination. In ρ-dependent termination, the features that cause pausing are unclear, and dissociation requires the presence of the ρ protein that employs a helicase activity to melt the DNA-RNA hybrid resulting in the dissociation of RNA polymerase.
Describe the mechanistic difference that distinguishes the splicing of group I introns from that of group II introns.
In the group I introns, the initial break in the RNA chain occurs as the 3'-hydroxyl group of a free guanine nucleotide not from the mRNA molecule makes a nucleophilic attack on the phosphodiester bond. In group II introns, the attacking species is the 2'-hydroxyl group of an adenylate residue in the intron itself.
What is the difference between ρ dependent and independent?
Independent uses weakly bonded A-U region and hairpin. Dependent uses helicase activity.
What are cyclobutane thymine dimers created by?
Induced by UV radiation. Generates a block to replication.
What stage(s) of protein synthesis does IF-2 interact with?
Initiation
What stage(s) of protein synthesis does the Shine-Dalgarno sequence interact with?
Initiation
What stage(s) of protein synthesis does GTP interact with?
Initiation and elongation
What is the translation cycle?
Initiation, Elongation, Termination
What is primase?
Is an RNA polymerase
What is DNA ligase?
Joins lagging strand pieces to each other
What enzyme is used to convert NMPs to NTPs?
Kinases. Using ATP
How did Meselson and Stahl determine that DNA replication is semiconservative?
Label parent DNA with 15N and daughter molecules show hybrid DNA (1st gen) and second gen shows 2 light DNA (14N) and 2 hybrid DNA
What does exinuclease do?
Makes 2 cuts in nucleotide excision repair and excises the damaged DNA.
What are the four major mechanisms of DNA repair?
Mismatch repair, base excision repair, nucleotide excision repair, direct repair.
What happens first in base excision repair and what's the enzyme?
N-glycosyl bond cleaved by DNA glycosylase
DNA Ligase requires what cofactor?
NAD⁺
If DNA underwound by 2 turns then what is the ΔLK?
Negative (-2). LK specifies the number of helical turns in a closed circular DNA
What are introns?
Noncoding (Introns) mRNA sequences that removed during splicing.
What other type of splicing is Group II intron splicing similar to?
Nuclear mRNA splicing (also lariat intermediate)
Briefly describe the general strategy for nucleotide degradation.
Nucleotide degradation begins with the removal of the 5'-phosphate to generate the nucleoside. Next, ribose is removed to generate the free base.
What organisms use alternative RNA processing? Eukaryotes, prokaryotes, or both?
Only eukaryotes
What enzyme is targeted as a treatment for African Sleeping sickness?
Ornithine decarboxylase
What's the base precursor?
Orotate
What is the important cofactor in polyamine decarboxylation?
PLP
All DNA polymerases have 3' to 5' exonuclease activity but which Polymerase has 5' to 3' exonuclease activity as well?
Pol I
Which DNA polymerase replicates the genome (fastest one)?
Pol III
How is the polylinker region used in cloning of a plasmid?
Polylinkers have cut sites for a variety of restriction enzymes, allowing insertion of DNA fragments produced with any of them.
If DNA overwound by 2 turns then what is the ΔLK?
Positive (+2). LK specifies the number of helical turns in a closed circular DNA
list molecules that bind to each of the two regulatory sites of ribonucleotide reductase.
Primary site: ATP and dATP Specificity site: ATP, dATP, dGTP, dTTP
A molecule that specifically recognizes the desired piece of DNA in a large library is known as a what?
Probe
What is included in the transcription bubble?
RNA-DNA hybrid, DNA coding strand (single stranded)
What type of restriction site is CTGCA|G a sequence for?
PstI
What type of restriction site is G|ACGTC a sequence for?
PstI
How do the synthesis of purine nucleotides and the synthesis of pyrimidine nucleotides differ?
Purine synthesius starts with the formation of PRPP, whereas pyrimidines incorporatie the PRPP near the end of the pathway.
Describe the metabolic fate of degradation products derived from degradation of purine bases.
Purines are converted to uric acid and excreted
What's the major difference in the way purines and pyrimidines are assembled?
Purines: base is built stepwise onto the PRPP. Pyrimidines: precursor base added to PRPP.
rho dependent termination
RNA contains Rho binding site, rho binds and moves towards RNA polymerase, pulls transcript and template plate apart, release RNA, ending transcription
What is a ribozyme?
RNA molecule with catalytic activity, some are RNA enzymes.
What do σ³² promoters do?
Recognizes heat shock promoters
What do σ⁷⁰ promoters do?
Recognizes most E.coli promoters
What are introns?
Regions of genes (primarily eukaryotic) that in mRNA are transcribed but are not translated. They do not code for amino acid sequences within the protein that is coded by the gene. Thus they interrupt the colinearity between the nucleotide sequence of the gene and the amino acid sequence of the encoded protein.
What sequences are required in an expression vector (for use with E. coli) that are not essential in a cloning plasmid?
Regulated expression of the cloned gene requires a bacterial promoter and its associated operator; a transcription termination sequence; and a ribosomal binding site.
What is the purpose of TfR in RNA regulation?
Regulation of RNA Turnover. Prevents activity of RNase. multiple IREs in 3' UTR.
What is the purpose of ferritin in RNA regulation?
Regulation of translation, allows formation of 43S pre-initiation complex. Interacts with single IRE in 5' UTR.
What is the role of release factors in bacterial protein synthesis?
Release factors are proteins that bring about the release of the finished polypeptide when the ribosome encounters a termination codon in the mRNA.
What does DNA gyrase do?
Relieves the strain caused by unwinding
What is the general strategy for nucleotide degradation?
Removal of 5'-phosphate → nucleoside Removal of ribose → free base which can then be recycled by salvage pathways that combine free base directly with PRPP
What is 5'→3' exonuclease activity for? Which DNA polymerase can do this?
Removing RNA primers, Pol I
What would happen if we only have one origin or replication?
Replication would take too long.
Name one enzyme that is always used to make a cDNA library, but is generally not used to make a genomic DNA library.
Reverse Transcriptase
What is the function of reverse transcriptase?
Reverse transcriptase is used to make first a single-stranded DNA complementary to mRNA, then a double-stranded DNA
Explain the enzymatic reaction catalyzed by ribonucleotide reductase.
Ribonucleotide reductase converts ribonucleotides into deoxyribonucleotides
PRPP is the donor of what part of nucleotides?
Ribose-phosphate unit (all but the base and phosphates that are added by the kinase)
How is the single stranded DNA stabilized while the lagging strand is synthesized?
SSB
What are examples of deaminating agents?
Sodium nitrite (NaNO₂), sodium nitrate (NaNO₃), and nitrosamine. Al of these are nitrates.
What is DNA Pol III?
Synthesizes most of DNA
What does telomerase do?
Synthesizes the DNA in telomeres from an RNA template. It is a reverse transcriptase. The template is an RNA molecule that is a part of the enzyme. Telomerase is an RNP enzyme.
How do we stabilize the ends of eukaryotic chromosomes and how are these specialized structures synthesized?
Telomeres at the ends. Telomerase adds telomeres using an internal RNA template and has reverse transcriptase activity. Telomerase is a ribonucleoprotein. Enzyme can reposition and resume extension and proceeds 5' to 3' direction
What 3 things does DNA polymerase require?
Template strand, Primer with a 3' OH, and dNTPs
What stage(s) of protein synthesis does RF interact with?
Termination
What is the "closed loop model" for efficient translation?
The 40S ribosomal subunit has a elF4E section and a PAB section. The elF4E section adds the 5' cap and the PAB section adds the 3' poly(A) tail. The mRNA forms a closed loop in the process.
Explain the function of the two regulatory sites of ribonucleotide reductase.
The primary regulatory site controls overall enzymatic activity and the substrate specificity site controls relative enzymatic activity i.e., which substrates are preferentially reduced.
Why is there a lag in cell growth when bacteria are switched from a medium containing glucose to one containing lactose?
The lag represents the time required for lactose to enter the cell, be converted into the inducer allolactose, and turn on the synthesis of the genes of the lac operon that are essential for growth on lactose.
In prokaryotes such as E. coli, many operons that encode enzymes involved in amino acid biosynthesis begin with a sequence coding for a leader peptide. This peptide has no known enzymatic function and is rich in the amino acid that is synthesized by the enzymes coded for in the operon. What is the function of this leader peptide?
The leader peptide is integral to the regulatory mechanism called transcription attenuation. In prokaryotes, transcription and translation are closely coupled. The ability to translate the leader peptide signals that the relevant amino acid is readily available in the cell and that transcription of the operon is undesirable at this time. Translation through the leader peptide (when the amino acid is abundant) allows the formation of a hairpin attenuator structure downstream in the nascent RNA chain. This attenuator acts as a transcription terminator, causing dissociation of the RNA polymerase. When the relevant amino acid is in short supply, the ribosome stalls trying to synthesize the leader peptide. A different secondary structure forms in the nascent RNA, preventing formation of the terminator and allowing continued transcription of the operon.
why does a ribonucleotide reductase require two regulatory sites instead of one?
The primary regulatory site controls the overall activity (energy charge and product feedback). The substrate specificity site works to provide a balanced pool of nucleotides.
When a mismatch is detected which strand gets degraded? How does it know?
The newly replicated strand, because it doesn't have any methyl groups.
Describe the metabolic fate of degradation products derived from degradation of pyrimidine bases.
The nitrogen atoms of pyrimidines are converted to urea (excreted); the carbon backbones enter the citric acid cycle.
In contrast to bacteria, eukaryotic chromosomes need multiple DNA replication origins. Why?
Their replication rate is much slower, and it would take too long with only a single origin per chromosome.
Certain restriction enzymes produce cohesive (sticky) ends. What does this mean?
They make a staggered double-strand cut, leaving ends with a few nucleotides of single stranded DNA protruding.
Certain restriction enzymes produce cohesive (sticky) ends. What does this mean?
They make a staggered double-strand cut, leaving ends with a few nucleotides of single-stranded DNA protruding.
How do snoRNAs guide cleavage and nucleoside modification of rRNA precursors?
Through base-pairing interactions.
Upon termination the chromosomes are intertwined. How are they separated?
Topoisomerase IV
Define topoisomerase, and explain the difference between type I and type II topoisomerases.
Topoisomerases are enzymes that change the linking number in a closed-circular, double-stranded DNA molecule by breaking one or two strands, adding or removing twists, and rejoining the strand(s). Type 1 topoisomerases break and rejoin only one strand, changing the linking number in increments of 1. Type 2 topoisomerases break and rejoin both strands, changing the linking number in increments of 2.
transactivators
bind to DNA at enhancer sites, interact w coactivators to activate transcription
PRPP is an intermediate in the synthesis of what three things?
Trp, His, Nucleotides
True or false? A lariat intermediate is formed during mRNA splicing.
True
True or false? DNA polymerase I possesses 5' → 3' exonuclease activity
True
True or false? Most regulation is positive (involves activators, not repressors) in eukaryotic gene expression regulation.
True
True or false? RNA polymerase requires a DNA template and all four ribonucleoside triphosphates.
True
True or false? RNA polymerase synthesizes an RNA product in the 5'→3' direction.
True
True or false? The 5' terminal cap structure of eukaryotic mRNAs is joined to the mRNA by a 5'-5' triphosphate linkage.
True
True or false? The polymerization of DNA is essentially irreversible due to the hydrolysis of pyrophosphate.
True
True or false? The purpose of SSB is to prevent single-stranded DNA from folding back on itself to form double-helical regions.
True
True or false? Transcription is associated with changes in chromatin structure in eukaryotic gene expression regulation.
True
How is serotonin made?
Tryptophan is hydroxylated to form 5-hydroxytryptophan, which is decarboxylated to form serotonin.
The double helix as determined by Watson and Crick is composed of what?
Two antiparallel complementary strands
What is DnaB helicase?
Unwinds DNA
What is the mechanism behind E. coli's preference for glucose over lactose?
When [glucose] is high, cAMP is low and CRP does not bind to the lac operon to stimulate transcription. (When [glucose] is low, cAMP is high and CRP-cAMP binds to the lac operon to stimulate transcription.)
When the growth medium contains both lactose and glucose, what proteins will be bound to the lac operon regulatory region?
When both glucose and lactose are present, neither lac repressor nor CRP will be bound.
basal transcription factors
bind to promoter region and recruit RNA polymerase to promoter
Outline one of the experimental methods providing evidence that the genetic code was a triplet code.
When one or two nucleotides were added to or deleted from a gene, the resulting mRNA produced a protein with a different amino acid sequence after the deletion or insertion. When three nucleotides were added or deleted, the resulting protein had a normal sequence except for the insertion or deletion of a single amino acid residue.
repressor
acts to impede access of RNA Polymerase to promoters
Which amino acids can recognize a C-G base pair?
arginine
What are the precursors for the purine ring?
aspartate, CO₂, glycine, formate, Amide N of glutamate
BAC
bacterial artificial chromosome, DNA construct used for transforming/cloning bacteria
What is the structure of a nucleoside?
base + sugar
What is the structure of a nucleotide?
base + sugar + phosphate
Which repair pathway is most likely to repair deamination?
base excision repair pathway
Which DNA binding motif is made of amphipathic helices (dimerization) and basic residues (DNA binding)?
basic helix-loop-helix
What are histones?
basic proteins that are usually associated with DNA?
histones
basic proteins, associated w DNA
Prokaryotic DNA replication proceeds in two directions, and is called what?
bidirectional replication
Name some elements of RNA secondary structure.
bulge - one mis/unmatched base internal loop - multiple mismatched bases hairpin - loop at end of complementary sequences
What are the precursors for the pyrimidine ring?
carbamoyl phosphate and aspartate
topoisomerases
change linking number
What is the order of events in the initiation of transcription in E. coli?
closed complex, open complex, start of RNA synthesis, promoter clearance
In the end of transcription the RNA transcript looks like which DNA strand?
coding strand, except with uracil.
plasmid
convenient cloning vector with which to introduce foreign DNA into E Coli
mismatch repair
corrects replication errors, parental strand is methylated, modify new strand
What are the pyrimidine nucleobases?
cytosine, thymine (DNA), uracil (RNA)
pyrimidine bases
cytosine, thymine, uracil
How are biological amines derived from amino acid precursors?
decarboxylation
What is the first step in polyamine synthesis?
decarboxylation
What is the key reaction in the synthesis of biological amines?
decarboxylation of amino acids
E. coli cells are placed in a growth medium containing lactose. How would a Lac repressor mutation that prevents dissociation of Lac repressor from the operator affect the expression of the lactose operon?
decrease in expression
E. coli cells are placed in a growth medium containing lactose. How would a mutation that inactivates b-galactosidase affect the expression of the lactose operon?
decrease in expression
E. coli cells are placed in a growth medium containing lactose. How would a mutation that inactivates galactoside permease affect the expression of the lactose operon?
decrease in expression
E. coli cells are placed in a growth medium containing lactose. How would a mutation that prevents binding of CRP to its binding site near the lac promoter affect the expression of the lactose operon?
decrease in expression
E. coli cells are placed in a growth medium containing lactose. How would the addition of high levels of glucose affect the expression of the lactose operon?
decrease in expression
Define "repression", with respect to regulation of transcription.
decreased transcription
What is the difference between deoxyribonucleotides and ribonucleotides?
deoxyribonucleotides lack a hydroxyl group at the 2' carbon on the ribose sugar.
phosphate ester
diff between nucleoside and nucleotide
What is the role of a phosphate ester bond?
difference between a nucleoside and a nucleotide
Is lagging strand replication continuous, discontinuous, or semidiscontinuous?
discontinuous (and opposite to fork movement)
What biological amine(s) are made from tyrosine?
dopa, dopamine, norepinephrine, epinephrine
When the bacterium E. coli is supplied with both lactose and glucose, which of the sugars is metabolized preferentially?
glucose
What are tetrapyrroles derived from in plants?
glutamate
How is γ-aminobutyrate (GABA) made?
glutamate is decarboxylated to from γ-aminobutyrate (GABA)
Which amino acids can recognize a T-A base pair?
glutamine or asparagine
What are tetrapyrroles derived from in animals?
glycine and succinyl-CoA
plasmid PBR322
has located recognition sites for restriction enzymes, replication origin which permits autonomous replication, resistance to more than 2 antibiotics, permits rapid screening for recombinant plasmids containing foreign DNA, small overall size
Which DNA binding motif is made of a recognition helix positioned in the major groove?
helix-turn-helix
Shine Dalgarno Sequence
helps recruit ribosome to mRNA to initiate protein synthesis
δ-Aminolevulinic acid is an intermediate in the biosynthesis of what?
heme
What biological amine(s) are made from histidine?
histamine
How is histamine made?
histidine is decarboxylated to form histamine
rho independent
hits G/C rich region, fold back on itself, G binds C, results in hairpin - string of Us bind As, weak bonds produce instability for enzyme to fall off
Which DNA binding motif is a widely conserved motif of 60 amino acids?
homeodomain
To determine the Tm for a solution, you measure the absorbance of light as a function of increasing temperature. Does the absorbance increase or decrease as the temperature is raised?
increase
Define "induction", with respect to regulation of transcription.
increased transcription
IF2
initiation phase, binds to initiator tRNA and controls entry of fMET-tRNA to ribosome
leader peptide
integral to transcription regulation - translation of leader peptide signals AAs available, forms attenuator - stops transcription - RNA pol dissociates. when AAs not available - can't synthesize leader peptide - transcription continues
What is the leading strand?
is synthesized continuously.
What is the role of a phosphodiester bond?
joins adjacent nucleotides in one strand
N- glycosidic
joins adjacent nucleotides in strand
hydrogen bond
joins complementary nucleotides in strands
What is the role of a hydrogen bond?
joins complementary nucleotides in two strands
At the replication fork, which strand is synthesized discontinuously?
lagging strand
What type of damage to DNA would nucleotide excision repair be used to fix?
large DNA lesions (i.e. from UV)
At the replication fork, which strand is synthesized continuously?
leading strand
Which DNA binding motif is made of amphipathic alpha helices with hydrophobic amino acid residues?
leucine zipper
phosphodiester bond
links 3' and 5' ends
What is the role of a N-glycosidic bond?
links base to pentose in nucleotide
DNA ligase
links okazaki fragments, joins lagging strands
What did the Khodursky and Bernstein experiment in '03?
mRNA Levels are regulated through both synthesis and decay. Steady state mRNA was moving through a test tube. When transcription was greater than degradation, the level in the tube went up. When degradation was greater than transcription, the level in the tube went down.
What are potential points of gene regulation in eukaryotes?
mRNA degradation and translation
coding strand
matches mRNA except T--> U
coactivator
mediates interactions between RNA polymerase and transactivators - does NOT directly bind DNA- ex. TFIID and Mediator: involved in activation when bound to transactivator where they help recruit basal transcriptional machinery to RNAP II to promoter
probe
molecule that recognizes desired piece of DNA in large library
Which enzymes catalyze the phosphorylation of dADP, dGDP and dCDP?
nucleoside diphosphate kinases
How can the linking number (Lk) of a closed-circular DNA molecule be changed?
only by breaking one or both strands
What is the name for the site in the DNA where DNA replication in E. coli begins?
origin
Difluoromethylornithine (DMFO) is an inhibitor of what enzyme?
ornithine decarboxylase
The enzyme converting ornithine to putrescine in the polyamine biosynthesis pathway is called what?
ornithine decarboxylase
Explain briefly the properties of the plasmid pBR322 that make it so convenient as a vector for cloning fragments of foreign DNA.
pBR322 has two antibiotic resistance markers, so that its presence in a bacterium can be detected, bacteria that carry it can be selected, and insertion of cloned DNA into one of the resistance markers can be detected by loss of antibiotic resistance. The plasmid also has an origin of replication, so that it replicates autonomously in E. coli. Several convenient restriction sites allow easy insertion of restricted DNA fragments. Its small size facilitates its entry into E. coli by standard transformation protocols.
Are prokaryotes monocistronic or polycistronic?
polycistronic
fusidic acid
preventing translocation of mRNA from A to P site catalyzed by EF-G-GTP (elongation step)
SSB
prevents reannealing of DNA at replication fork
On the strand synthesized discontinuously, the short pieces called Okazaki fragments are initiated by an enzyme that synthesizes an RNA primer and is called what?
primase
What enzyme adds RNA primers?
primase
What is the 3'→5' exonuclease activity of DNA polymerase for?
proofreading
What type(s) of RNA does Pol I transcribe?
rRNA
ribozyme
ran molecules, cleave phosphodiester bonds, transesterification, post transcriptional processing
What does topoisomerase I do?
relaxes negatively supercoiled DNA
DNA gyrase
relieves buildup of positive supercoils
DNA pol I
remove primers, seal nicks, fill in gaps , has 5--> 3 exonuclease activity
base excision repair
removes damaged base, damaged base has different DNA glycosylase enzyme for each base lesion, which cleaves N glycosyl bond, removing base
What type of damage to DNA would mismatch repair be used to fix?
replication errors
When a DNA molecule is described as replicating bidirectionally, that means that it has two of what?
replication forks
DNA B (helices)
required for unwinding DNA
puromyocin
resembles aminoacyl terminus of aminoacyl tRNA and binds to A site
peptidyl transferase
ribozyme in 50s ribosomal subunit- catalyzes formation of peptide bond as ribosome moves along mRNA
How much of the human genome is translated into proteins?
roughly 1.5%.
Is DNA replication continuous, discontinuous, or semidiscontinuous?
semidiscontinuous
What biological amine(s) are made from tryptophan?
serotonin
operon
set of genes and regulatory DNA sequence for that set of genes
inducer
small molecule that binds activator and induces transcription
vector
small piece of DNA taken from plasmid, can be maintained in organism, foreign DNA fragment can be inserted for cloning
What small molecules are active in the spliceosome in order to make mRNA splicing work?
snRNPs
Vertebrate rRNA uses what type of small RNA molecule for processing?
snoRNAs
Ornithine decarboxylase is involved in the synthesis of what polyamines?
spermine and spermidine
What does SSB do?
stabilizes the single stranded DNA
The technique known as two hybrid analysis for detecting interacting gene products depends on what?
stimulation of transcription by interaction of two Gal4p domains via fused protein sequences.
How do cells store iron?
stored in the protein ferritin
δ-Aminolevulinic acid is formed from what?
succinyl-CoA and glycine
What is the lagging strand?
synthesis direction is opposite to that of fork movement. Is synthesized discontinuously.
DNA pol III
synthesizes most DNA for replication including synthesizes lagging strand
primase
synthesizes primer, necessary for replacement of primers,
What type(s) of RNA does Pol III transcribe?
tRNA, 5S rRNA
What is the role of tRNA^fMet in bacterial protein synthesis?
tRNA^fMet is the transfer RNA that initiates protein synthesis by inserting the first amino acid (fMet) in every prokaryotic protein.
The RNA transcript is made by pairing to which DNA strand?
template
given mRNA sequence might be translated in any of three reading frames. Describe how prokaryotes determine the correct reading frame.
the Shine-Dalgarno sequence in the mRNA base pairs with a complementary sequence in the 16S RNA of the ribosome; this positions the correct start codon (AUG) on the 30S ribosomal subunit. Thus, the initiating AUG is distinguished by its proximity to the Shine-Dalgarno sequence.
given mRNA sequence might be translated in any of three reading frames. Describe how eukaryotes determine the correct reading frame.
the initiating AUG codon is the first AUG that the ribosome encounters as it scans the mRNA from its 5' end. In both cases the initiating AUG also sets the correct reading frame.
What eukaryotic regulatory proteins interact with enhancers?
transactivators
introns
transcribed not translated, spliced out before translation
sigma 70
transcription initiation factor that enables specific binding of RNA polymerase to gene promoters
The hormones epinephrine and norepinephrine are derived biosynthetically from what amino acid?
tyrosine
How is dopamine made?
tyrosine is hydroxylated and the product (dopa) is decarboxylated to form dopamine.
What is the linking number of a relaxed closed-circular DNA molecule with 5,000 base pairs with a break in one strand? Assume that the DNA is a right-handed helix with 10 base pairs per turn, and that one round of reaction equals one turnover.
undefined
What does DnaB helicase do?
unwinds the DNA
An intermediate of purine degradation in humans is what?
uric acid
nucleotide excision repair
used to remove large bulky lesions- more than 1 base affected - uses excinuclease, DNA Pol 1, DNA ligase
How are introns removed?
via splicing
Describe the mechanism by which the leader-attenuator region fine tunes the extent of transcription of the structural genes in the trp operon.
when Trp high, the ribosome covers sequence 2 preventing 2/3 base pairing but causing the formation of a rho independent termination structure which includes both a stem loop and poly U causing premature termination of RNA polymerase. when trp low, ribosome slows down at Trp codons in leader peptide leading to sequence 2-3 base pairing. Sequence 3 is not available for base pairing with 4 and no rho independent terminator forms and there is no premature chain termination. The trp structural genes are then transcribed.
Which DNA binding motif is made of coordinated Zn²⁺ to cysteine residues?
zinc finger
What biological amine(s) are made from glutamate?
γ-aminobutyrate (GABA)
What is the subunit in E. coli RNA polymerase that is responsible for recognition of the promoter?
σ