Exam 1 Flashcards
(164 cards)
1
Q
Benefit of classical genetics
A
Allows characterization of mutants without knowledge of molecular basis
2
Q
Benefit of molecular genetics
A
Allows isolation and mutation of a gene without knowing its function
3
Q
What are two big benefits of working with bacteria?
A
Haploid so no recessive genes. All progeny genetically identical so easy to get purebred line
4
Q
What are distinguishing features of eukaryotes?
A
Nuclear membrane decoupling txn and tln. Other membrane bound organelles
5
Q
Research contribution of Griffiths
A
DNA as transforming principle. Rough-no dz. smooth-dz. rough + killed smooth - dz.
6
Q
Research contributions of Chargaff.
A
A:T = C:G = 1:1 in all species
7
Q
What is the difference between a nucleotide and a nucleoside?
A
Nucleotide has phosphate, nucleoside does not.
8
Q
Describe a purine. Which nucleotides are purines?
A
9-membered, 2-ring structure. A and G
9
Q
Describe a pyrimidine and which NT are pyrimidines
A
Single 6-membered ring. C, T, and U.
10
Q
How does labeling of nitrogenous bases and sugar bases differ?
A
Sugars label carbon only and use prime suffix. Nitrogenous bases label carbon and nitrogen and do not use prime suffix.
11
Q
Describe the polarity of nuclei acids
A
5’ end = phosphate = upstream
3’ end = OH group = downstream
12
Q
Describe polarity of proteins
A
N terminus is amino end. C terminus is carboxyl end.
13
Q
What is the central dogma?
A
DNA > transcription > RNA > translation > protein
14
Q
Describe cis acting sequence and trans acting factor in relation to chromosomes.
A
Cis is the sequence that interacts with a protein. Trans acting factor is the protein.
15
Q
Describe cis acting sequence and trans acting factor in relation to gene structure
A
Cis acting sequence is the ribosome binding site. The trans acting factor is the ribosome.
16
Q
Why is polarity important?
A
Polarity allows genes to be read in the correct direction.
17
Q
What is the downfall of linear chromosomes and how do eukaryotes deal with it?
A
End replication problem. Telomeres and telomerase.
18
Q
How do bacteria compact DNA for storage?
A
They have no histones but use histone like proteins (HU, HN-F, Fis, IHF)
19
Q
Where are chromosomes localized in bacteria? Why?
A
Nucleoid. Allows colocalization of transcription and translation.
20
Q
What is semi-conservative replication?
A
2 strands are replicated. Each daughter cell receives one old and one new strand.
21
Q
Where does active replication occur?
A
Replication fork
22
Q
Where is replication initiated?
A
Origin of replication (ORI)
23
Q
What is the role of ribonucleotide reductase (RNR)?
A
NDP > dNDP
24
Q
What is the role of kinase in producing dNTPs?
A
dNDP > dNTP
25
What is the role of thymidylate synthase?
dUMP > dTMP
26
What is the role of DNA polymerase I?
Replication, gap filling to connect Okazaki fragments, DNA repair
27
What is the role of DNA polymerase III?
Replicates majority of chromosome
28
What DNA polymerases in E. coli only repair DNA?
II, IV, V
29
What is the direction of synthesis of DNA strand during replication? Reading of template strand?
Synthesis 5' to 3'. Reading template 3' to 5'.
30
Are there any 3' to 5' DNA polymerases?
No. Okazaki figured out why.
31
Can DNA polymerase initiate DNA synthesis?
No. Only RNA poly can.
32
What does Primase do? What kind of enzyme is it?
Synthesizes RNA primer that DNA polymerase needs to initiate. It is an RNA polymerase.
33
What is a benefit of priming for DNA replication with RNA?
Prevents mistakes because if a mistake is made, the RNA in a RNA/DNA hybrid will always be replaced by DNA. To repair DNA/DNA hybrids, a long strand is necessary, so mistakes would be less likely to be repaired in a short primer
34
Which DNA polymerase has nick translation activity (5'-3')
DNA polymerase I
35
What is the role of DNA ligase?
Catalyzes phosphodiester bond between 5' P and 3' OH. Allows connection between Okazaki fragments.
36
What is the role of the sliding clamp?
Tethers DNA polymerase to the template.
37
What is processivity?
How long a DNA polymerase stays on the DNA. It is highly influenced by the ability to bind to sliding clamp.
38
What does DNA helicase do? What is another name for it?
Aka DNAb. ATP dependent separation of two parental DNA strands during replication
39
What are SSBs and what do they do?
Single strand DNA binding proteins. (Helix destabilizing proteins) maintain separation of strands after DNAb activity by binding to separated strands without obscuring exposed bases.
40
Topoisomerases
Relieves stress of supercoiling as the double helix is unwound. Topoisomerase I breaks one strand while topoisomerase II breaks two strands.
41
Role of condensins in replication
Catalyze chromosome condensation and prevent daughter chromosomes from becoming intertwined.
42
How large is the lag between leading and lagging strand during replication?
2kb
43
Where are Okazaki fragments and what are they?
On lagging strand during replication. They are DNA fragments extended from RNA primers.
44
What two enzymes are important in lagging strand synthesis that are not needed on the leading strand?
DNA polymerase I (5' to 3' exonuclease activity) to remove RNA primer and refill with DNA and DNA ligase to join Okazaki fragments.
45
What is the role of 5'-3' vs 3'-5' exonuclease activity?
5'-3' primer removal
| 3'-5' proofreading
46
What is the klenow fragment?
The large domain of DNA polymerase I which retains 3'-5' activity (proofreading) but loses 5'-3' activity (primer removal).
47
Where is the 3'-5' activity of DNA polymerase III?
In an accessory protein (epsilon subunit)
48
Name four impediments to DNA replication
Proteins associated with chromosome (rna pol)
Nicks in template strand
Supercoiling
Bulky mutations
49
What is methyl-directed mismatch repair
It is a backup repair mechanism. Old DNA is fully methylated, so the partially methylated strand is repaired where it doesn't match the fully methylated strand.
50
Name two methods to deal with impediments to DNA replication
Re-priming of DNA synthesis downstream from the lesion
| Displacement of RNA Pol and re-initiation of DNA replication using residual mRNA as primer
51
What are some characteristics of oriC?
It is a cis-acting site.
It is AT righ for bi-directional initiation --> two replication forks
COntains sites for trans-acting factors of initiation (including DnaA)
52
What does DnaA do?
IT sets up the origin by allowing the following:
Helicase (DnaB/C) to unwind helix
SSB to keep the strans apart
Primase (DnaG to make RNA primers)
53
What are some characteristics of ter?
It is the terminus
Signals termination of replication
terA and terB are unidirectional sites of the opposing replication forks
54
Why do ter sites require accessory proteins?
They need accessory proteins to impede the progress of helicase. Two examples are:
tus: terminus utilization substance
RTP: replication terminator protein
55
What is the Xer Recombination system
It makes sure that the correct copy of replicated DNA gets to the new cell without crossovers.
56
What system allows formation of dimers, which does not?
General recomobination --> creation of dimers
| Xer Recombination system --> Resolution of dimers
57
What are the cis-acting and trans-acting components of the Xer recombination system?
cis-acting: dif (targets of XerCD), KOPS (recognized by FtsK)
Trans-acting: Xer recombinase (XerCD), Ftsk (a polar DNA translocase)
58
What is required for XerCD activity?
Ftsk chromosome positioning system and TWO dif sites on the SAME DNA molecule
59
What is the role of FtsK DNA Translocase?
It is used to pump and distribute replicated chromosomes into individual daughter cells. This is an energy dependent process (Uses ATP)
60
What is the role of KOPS in the Xer Recombination system?
they orient the activity of the FtsK translocase so that the dif sites can be moved to the septum (the site of cell division)
61
What are catenanes?
Interlinked chromosomes that can form due to replication
62
How does decatenation proceed?
Toposiomerase IV cleaves both strands of one molecule, passes the other molecule through the break, and reseals the double stranded break.
63
What role does FtsZ play in replication?
IT is a protein related to tubulins that acts in cytokinesis. It acts as a scaffold for the separation of proteins at the septum.
64
What is the role of Min proteins in replication?
It ensures the septum (the site of cell division) forms in the middle of the cell during cytokenisis
65
What is the role of Nucleoid Occlusion Proteins (Noc) in replication?
They prevent FtsZ formation when nucleoid is in the center of the cell during cytokinesis.
66
What is synthetic lethality. Give an example from replication.
It is when KO of one or the other of two genes is ok, but KO of both is lethal. Eg. Min and Noc proteins involved in cytokenisis.
67
How is methylation status of DNA involved in regulation of cell cycle division?
SeqA is a negative regulatior of replication, which recognizes hemi-methylated GATC sites and sequesters hemi-methylated oriC --> ensures replicates once per chromosome per cell cycle.
68
How many sense codons are present in the genetic code?
61
69
How many stop or non-sense codons are there? What are they?
3 - UAG, UAA, UGA
70
What is the initiation codon and what amino acid does it specify?
AUG - codes for fMET
71
How many levels of organization are present in protein structure? Describe them.
1 - AA sequence
2 - alpha helices and beta barrels
3 - interactions between charged areas of protein chain
4 - multi subunit interactions
72
What is the difference in abundance and half life between mRNA, tRNA, and rRNA?
mRNA is least abundant and has shortest half life. tRNA and rRNA are abundant and have long half lives.
73
What levels of organization are there in RNA molecules?
Primary, secondary (hairpins), and tertiary (pseudoknots)
74
What post -transcriptional changes can occur in RNA?
Processing - cleavage and addition of nucleotides
| Modification - enzymatic modification of bases
75
Describe the structure of RNA polymerase
It is a multi-subunit enzyme with a core enzyme and a holoenzyme plus a sigma factor.
76
What RNA does bacterial RNA polymerase NOT make?
primers for replication
77
What is the direction of synthesis? The direction of DNA template reading?
Synthesis is 5' to 3'
| Reading is 3' to 5'
78
How does initiation of transcription differ from initiation of replication?
No need for a primer or helicase for replication
79
What is the role of Promoters in transcription and what is the first base?
They dictate the transcription initiation site (known as +1). by RNA Pol. It is usually a pyrimidine codon, so the first base of RNA is usually a purine.
80
What is the role of sigma factors?
THey determine which genes are to be transcribed. for example E. coli sigma 70 initiates transcription of housekeeping genes.
81
How do sigma factors recognize initiation sites.
Promoters recognized by teh same sigma exhibit a consensus sequence. Eg. Sigma 70 promoters have a characteristic -10 and -35 region. They can see it in closed DNA by reading major groove.
82
What are teh 3 channels in a transcribing RNA Pol
Secondary site channel: NTP entry
Active site channel: Phosphodiester bond formation
Exit channel: Transcript exit from enzyme.
83
Describe transcription initiation
Sigma recognizes promoter. Domain 2 of sigma unwinds DS DNA --> open coplex. Transcription bubbble forms and DNA-RNA hybrid (~10nt) is formed. SOon after initiation, sigma is released from the holoenzyme. Only the core of RNA Pol participates in elongation. Purine enters secondary channel. When second NT forms first phosphodiester bond, initiation complex is established.
84
Describe transcription elongation
the active site is ~10nt. Transcript emerges at ~50nt/sec. There is frequent pausing and backtracking., which is probably due to secondary structure of RNA as it leaves the complex.
85
Describe Factor-independent terminators of transcription
Function is dependent on secondary RNA structure. An inverted repeat sequence and POly A --> RNA Hairpin
86
Describe Factor dependent terminators of transcription
No identifiable sequence features. Function is dependent on a termination factor (rho), which is a DNA-RNA helicase, which unwinds the DNA-RNA hybrid. IT requires that the RNA is not being translated.. Requires a rho-sensitive pause site. Rho binds to rut site on transcript.
87
WHat is the cis acting sequence and trans acting factor in factor dependent termination of transcription?
Cis acting sequence is rho-sensitive pause site.
| Trans-acting factor is Rho.
88
What post-transcription processing occurs in bacterial mRNA
None
89
WHat happens post-transcriptionally to rRNA and tRNA?
they are transcribed as a single precursor, with spacers betwene genes. Individual rRNAs and tRNAs are derived via processing of primary transcript by RNases.
90
Know figure 2.28
Just know it!
91
What it wobble base-pairing?
the same tRNA (anti-codon) can pair with more than one codon (on mRNA)
92
What is redundancy in translation? When does it not apply?
More than one codon specifies most amino acids. Exceptions are TRP and MET.
93
Which end of the protein emerges from the ribosome first?
N-terminus
94
What is the primary energy source for translation?
GTP
95
What is the start codon?
AUG most commonly
96
What makes fMet unique
It is structurally similar to a peptidyl-tRNA rather than amino-acyl-tRNA, allowing it to occupy the P site.
97
What happens to fMet post-translationally?
It is usually removed.
98
What is the shine-dalgarno sequence? What is the term when it is not present?
It is a sequence ~5-10 nt upstream of initiation codon and is complimentary to the 3' end of the 16s rRNA (component of 30S subunit). Those without S-D sequence are leaderless mRNAs
99
What is the role of IF1 in initiation of translation?
It binds to A site, preventing tRNA-fMet from binding here rather than P site.
100
What is the role of IF2 in initiation of translation?
Binds tRNA-fMet and GTP and localizes it to the P site. Aids recruitment of the 50s subunit.
101
What is the role of IF3 in initiation of translation?
plays a role in keeping ribosome subunits apart. Prevents binding of large subunit.
102
What direction does the ribosome move on mRNA template?
5' to 3' one codon at at time.
103
Describe the reaction of translation elongation.
- aa-tRNA associated with EF-Tu-GTP delivered to A site
- Accurate codon-anti-codon pairing --> GTP hydrolysis and release of EF-Tu-GDP.
- ET-Ts catalyzes reconversion of EF-Tu-GDP to EF-Tu-GTP
104
What reaction does peptidyl transferase catalyze?
Transpeptidation. the formation of the peptide bond via transfer of growing polypeptide chain at the P site to amino group of the aa-tRNA in the A site.
105
What is peptidyl transferase made of?
It is a ribozyme. it is part of the 23s rRNA
106
What prevents initiator aa-tRNA from occupying the A site?
the formyl group on fMet and IF1
107
What does translocase (EF-G) do?
it moves the ribosome 3 nt downstream towards 3' end of mRNA template.
108
Describe how translocase acts.
- Uses GTP as an energy source.
- naked tRNA from P site, now in E site and can exit.
- Polypeptide-bearing peptidyl-tRNA in A moves to P site
- A site is now empty and ready to accept another aa-tRNA.
- The ribosome ratchets 50s then 30s, rather than moving as a whole.
109
What is required for termination of translation?
Stop codon (UAA, UGA, UAG), Release factor proteins
110
What are three characteristics of release factor proteins in translation?
Recognize stop codons
Catalyze polypeptide relase from late peptidyl-tRNA
Structurally similar to aa-tRNA (molecular mimicry)
111
What post-translation modification is done to the polypeptide?
Deformylation of fMet and removal of the N-terminal Met.
112
When does ribosome rescue occur?
When ribosome is translating a truncated mRNA (with no stop codon before 3' end of mRNA
113
What is tmRNA?
RNA that has both tRNA (has aa Ala attached to acceptor arm) and mRNA (has an ORF w/ stop codon) features.
114
Describe ribosome rescue
- Transpeptidation of polypeptide to Ala of TmRNA at A site.
- translation of tmRNA ORF until stop codon
- the tmRNA codes a peptide that signals degradation of the entire rescued (and defective) protein.
115
What features do polycistronic mRNAs have?
each gene is separated by an inter-cistronic spacer and has its own TIR (S-D box and initiation codon)
116
What is translational coupling?
Translation of downstream gene is dependent on translationof upstream gene (see fig 2.40). REMEMBER - transcription of downstream gene is NOT affected (only translation)
117
What is transcriptional polarity?
may be due to insertion that introduces a factor independent transcription terminator or a nonsense mutation in an upstream gene that exposes a downstream factor dependent terminator. REMEMBER Transcription of the downstream gene IS prevented.
118
Wild-type
THe archetype of the species
119
Mutant
Strain harboring a genetic difference from wildtype
120
Variant/Strain
Slight genetic difference from progenitor
121
Mutation
HERITABLE change in genome of organism. Not all mutations --> phenotpyic changes
122
Null Mutation
Complete gene knockout
123
Allele
Different version of the same gene. (Mutant and wild type are two alleles of the same gene)
124
Auxotroph
Requires essential nutrient from environement. DUe to mutation in biosynthetic pathway
125
Catabolic mutations
Prevent utilization of a particular nutrient. Due to mutation in catabolic pathways
126
Conditional lethal mutations
The lethal mutation is only expressed under certain conditions (restrictive/non-permissive) and is not expressed under permissive conditions. Useful in obtaining mutations in essential genes
127
Resistant mutation
a trait that can be easily selected for in a population (eg. amr)
128
Leaky mutations
Gene product retains partial function
129
Revertant
A mutant strain harboring an additional mutation that permits restoration of wildtype
130
Point mutations
single base-pair change
131
Transitions
Point mutation where same class of nucleotide substitutes (Pyr to Pyr or Pur to Pur)
132
Transversions
Pur to Pyr point mutations
133
Causes of point mutations
Chemical damage, irradiation, spontaneous changes
134
Spontaneous mutations
Replication of tautomeric forms of bases or incorporation of enol forms --> repari by proofreading activity of DNA Pol
135
Spontaneous deaminations
Conversion of C to U. Repaired by removal of U from its nucleotide --> abasic site which is then repaired
136
Missense mutations
substitution of one AA for another (may be conservative or non-conservative based on chemical character of AA)
137
Nonsense mutation
Introduces a stop codon --> truncated protein
138
Frameshift mutation
Due to insertion or deletion. MOst lead to premature termination of translation (early stop codon) --> null phenotype
139
Deletion mutation
Often results in null mutation or may result in gene fusions in coding region or regulatory regions.
140
Direct repeats
May occur on same or different DNA molecules. IF between different DNA molecules, there is a deletion in one and an insertion in the other. IF on same, loopin out allows deletion.
141
Tandem duplications
Created in teh process of making deletions. Unstable due to reversion. Play important role in creation of gene families. Rarely have different phenotype
142
Inversions
Due to recombination between inverted repeats on SAME DNA molecule. Order of genes between inverted repeats is reversed. Unstable due to re-inversion.
143
Programmed inversions
Site-specific recombination used to initiate inversions. Used in salmonella phase variation by promoter flipping due to inversion.
144
Insertion mutation
Usually due to a mobile genetic element. OFten results in frameshift and null mutations. This may exert transcriptional polarity on downstream genes in teh operon
145
Suppression of mutations
Restoration of function due to an additional second-site mutation
146
True reversion of mutation
Restoration of sequence to wild-type
147
Intragenic suppressors
Compensatory mutation in same gene
148
Intergenic/Extragenic suppression
Involves compensatory mutation in a different gene. May be second site mutation in another gene in teh same metabolic pathway
149
tRNA suppressor mutations
Mutation in anticodon of tRNA that allows base-pairing with stop-codon. USed to study stop codons. Produced polypeptide usually has a missense mutation. (Opposite of synthetic lethality) This mutated tRNA can be added in the lab to disrupt proteins.
150
Mutagens
Increase frequency of mutants in a population
151
How can you determine how many independent mutations exhibit the same phenotype
USe saturation mutagenesis. Use multiple cultures of WT, treat with different mutagens. Map mutants to determine how many genes it maps to.
152
Screening
Need to know expected phenotype. Growth of mutagenized population under non-selective conditions. Replica plate to identify clones that grow under permissive but not restrictive conditions
153
Selection
Condition that allows selective growth advantage for desired mutant --> more efficine than screens.
154
Recombination
Requires interaction and exchange between two DNA molecules. One from recipient, one from donor
155
General or homologous recombination
Occurs at all genomic sites between very similar DNA sequences
156
Site-specific recombination
Occurs at specific sties on DNA and dependent on enzymes dedicated to these sites
157
What is the result of linear DNA with one crossover?
Chromosome break (lethal in bacteria)
158
What is the result of linear DNA with two crossovers?
Reciprocal exchange of DNA
159
What is the result of circular DNA with one crossover?
Integration of donor into chromosome and duplication of homologous sequences.
160
What is result of circular DNA with two crossovers?
REciprocal exchange of DNA sequences between sites of recombination events.
161
What are suicide vectors?
Plasmids with temperature sensitive ORI that allows recombination in a temperature sensitive manner.
162
Marker rescue
Method to localize sites of mutation in short DNA sequences and/or functional domains in modular proteins.
163
Complementation
Interaction between diffusible products of genes on different DNA molecules.
164
Partial diploid
Strain harboring chromosomal mutated gene and WT copy of gene introduced via transformation, conjugation, or transduction.
