Exam 2 Flashcards
(156 cards)
1
Q
Where is prokaryotic DNA found?
A
Within the nucleoid
2
Q
Prokaryotic DNA is not…
A
confined by a membrane
3
Q
How does prokaryotic DNA fit inside the cell?
A
Supercoiling
4
Q
What is DNA?
A
polymer of dNTP’s
5
Q
4 dNTP’s
A
dATP, dTTP, dGTP, dCTP
6
Q
What does mature prokaryotic DNA look like?
A
Circular, don’t have ends
7
Q
How are DNA strands held together?
A
H bonds
8
Q
A-T
A
2 H bonds
9
Q
G-C
A
3 H bonds
10
Q
Purines
A
A and G
11
Q
Pyrimidines
A
C, U (found in RNA), T
12
Q
What makes DNA stable?
A
Double stranded character contributes to stability and integrity. Also does not have #2 OH group like RNA does, making it more stable than RNA
13
Q
What do replication intermediates look like?
A
Not circular yet, often do have ends
14
Q
Characterize RNA
A
Single stranded molecule, polymer of NTP’s (ribose), 5’ triphosphate end, 3’ OH end.
15
Q
RNA strands often have…
A
Complex folded structures Ex) tRNA
16
Q
Translocation direction
A
3’ to 5’
17
Q
Which molecule translocates across DNA?
A
DNA Polymerase
18
Q
Is DNA replication dependent on a template?
A
Yes - both parent strands have new daughter strands synthesized simultaneously
19
Q
What happens to the phosphates during DNA replication?
A
Pyrophosphates (Pi) are released and can be reused in other cellular processes. Pyrophosphate = beta and gamma phosphates (the ones not directly attached to 5’ carbon of incoming dNTP)
20
Q
What does DNA replication reaction rely on?
A
The cleavage of the bond between the alpha and beta phosphates - this allows for the addition of nucleotides and release of pyrophosphate
21
Q
How does this DNA replication reaction occur?
A
3’ OH (hydroxyl group) attacks the alpha phosphate of the incoming dNTP, causing release of pyrophosphate RNA polymerase basically catalyzes the same basic reaction (except uses NTP’s, not dNTP’s)
22
Q
How are dNTP’s held together in the DNA molecule?
A
Phosphodiester bonds (backbone becomes negatively charged due to the phosphate backbone being formed)
23
Q
How is the DNA replication reaction fueled?
A
The energy stored in the bond between alpha and beta phosphate group is released, which drives the enzymatic reaction forward.
24
Q
What is theta replication?
A
DNA synthesis occurring on a circular DNA molecule (since it looks like a theta); theta replication is a consequence of starting on a circular template
25
What two sites are important on the bacterial chromosome?
oriC (initiation) and ter (termination)
26
Where are oriC and oriT located?
Roughly 180º away from each other on the bacterial chromosome
27
Can initiation of DNA synthesis occur at different sites?
No: initiation always occurs at a single site on the bacterial chromosome (always at oriC)
28
Which direction is DNA synthesis occurring in on the bacterial circular chromosome?
Both directions --\> bidirectional
29
How many base pairs are in the oriC sequence?
Roughly 250 bp
30
How many replication forks form during theta replication?
2 replication forks since DNA synthesis is bidirectional
31
Where is 9-mer found in the DNA?
Found in oriC of bacterial chromosome; found in multiple copies; also called DnaA boxes
32
What happens when DnaA-ATP binds to DNA?
DnaA-ATP will bind to 9-mer/DNA box regions within the oriC region; multiple DnaA-ATP complexes will bind to DnaA boxes; when there are enough DNA-A ATP bound it distorts the A-T rich 13-mer sequence upstream of 9-mer sequence; distortion causes strands to separate and form single strands where other replication proteins can load onto DNA
33
What three components are critical for setting up the replication fork?
Primase, DNA helicase (DnaB) and DNA helicase loader (DnaC)
34
What is characteristic of DnaA boxes?
A-T rich regions with low amounts of H bonding that can easily be broken
35
Can DnaA-ATP hydrolyze ATP?
Yes; can hydrolyze ATP to ADP
36
Where is 13-mer located?
Within oriC; adjacent to DnaA-ATP binding sites
37
When is DnaA-ATP concentration lowest in the bacterial cell? When is highest?
DnaA-ATP is lowest directly after DNA replication; highest when cell is ready to replicate DNA and is ready to divide Timing of initiation is determined by [DnaA-ATP] (activated DnaA); has a very high affinity for DnaA boxes in oriC
38
Replication occurs at a replication fork using:
DNA pol (DNA pol III) Primase Helicase SSB
39
Where is DNA pol III found?
It is the main replication polymerase found in all bacteria.
40
What is the role of primase?
Primase synthesizes short RNA primers that are necessary during DNA replication; DNA pol III cannot catalyze reaction unless a free 3’OH is present, but RNA pol can —\> uses primase instead of RNA polymerase
41
Leading strand:
DNA synthesis is done continuously as the direction that DNA is separating allows DNA pol III to trans locate 3’ to 5’ as DNA is being melted
42
Lagging strand:
DNA synthesis is occurring discontinuously. 3’ to 5’ translocation is occurring in the opposite direction as the DNA is being unwound. Okazaki fragments (short patches of DNA) must be made as the DNA is unwound —\> requires multiple RNA primers laid down by Primase
43
What is the role of helicase?
Unwinds dsDNA further at the replication fork
44
What is SSB?
Single-strand Binding Protein —\> helps to stabilize the ssDNA and helps to keep the strands apart so that replication enzymes can act
45
Can DNA synthesis occur without RNA primers?
NO —\> RNA primers are necessary; at the beginning of each new strand, there will always be a short RNA primer that serves as the starting point for making a new DNA strand
46
How can we describe the symmetry of DNA strands at the replication form?
The process has ASYMMETRY; leads to different leading/lagging strand DNA pol III enzymes in some prokaryotes/many eukaryotes
47
Which strand does DNA Pol III synthesize? Leading or lagging?
BOTH —\> DNA Pol III is used for DNA synthesis in both leading and lagging strands in most bacteria
48
Why is the RNA primer used?
It provides a free 3’ OH group that DNA Pol III can use to attach a dNTP to start building nucleic acid
49
Can RNA polymerase initiate synthesis without an existing 3’OH end?
Yes! RNA Polymerases can take 2 NTP’s and attach them together to make the first phosphodiester bond in an RNA molecule In contrast, DNA Pol III cannot do this.
50
What is found at each replication fork?
2 copies of DNA Pol III 1 helicase 1 primase
51
Where do helicase and primase bind to after DNA is “kinked” by DnaA-ATP?
They bind to lagging strand template. They then enable the assembly of other proteins that have to accompany replication fork —\> forms active replication fork
52
What do clamp proteins (sliding clamp) do at the replication fork?
Tethers the 2 copies of DNA Pol III to each other
53
Okazaki fragment length
About 1000 bases long With an RNA primer initially at each 5’ end
54
How are RNA primers removed from Replicating DNA?
RNAse H (e-coli specific) or DNA pol I (some bacteria only use pol I) removes primers using exonuclease activity (degrades RNA until it reaches DNA) from RNA-DNA hybrid at the beginning of Okazaki fragments. DNA Pol I then replaces the RNA primer with DNA
55
What does DNA ligase do?
Final bond is made after replacing of RNA with DNA by DNA Pol I between 3’OH and 5’ phosphate
56
What is the rate of DNA replication?
750-1000 bases/sec
57
Wha makes replication accurate?
- Semiconservative replication (checked against parent strand) - fidelity of dNTP binding and 3’—\>5’ exonuclease (proofreading exonuclease found in DNA Pol I and III) - mistake rate is 1/10^9 nucleotides
58
What is the role of topoisomerases in bacteria?
Introduce super coils in the DNA to condense it in the nucleoid
59
What does DNA Gyrase do?
Reduces strain during replication
60
How does gyrase work?
It acts just ahead of the replication fork and enables the rep. Fork to keep unwinding and synthesizing new DNA on the 2 template strands - reduces increased strain downstream of unwinding so that unwinding continues to occur at a consistent rate
61
Another antibacterial drug target:
Topoisomerases
62
Where does DNA replication end?
At terminus (opposite end of circular chromosome)
63
What is found within terminus region?
15-30 base pair terminator sequence (ter)
64
Are the chromosomes separated directly after replication?
No - they are linked rings called catenanes.
65
How does DNA synthesis stop when replication forks meet?
Tus protein binds to ter sequences and acts as an orientation-specific block for replication —\> acts as helicase antagonist
66
How are catenanes separated?
- Topoisomerase IV - site-specific recombinase (such as XerC and XerD)
67
Replication forks will be blocked from...
Replicating past Tus-ter complexes. - arrests replication fork progression and preventing endless replication cycles on circular molecule
68
How does Tus interact with helicase?
Tus antagonizes helicase involved with the replication fork and prevents it from proceeding through
69
DNA Cell composition dry weight (%)
3% (10-20 DNA Pol III/cell)
70
RNA Cell composition dry weight (%)
20.5% RNA - 16.4% rRNA (lots of ribosomes) - 2.5% tRNA - 1.6% mRNA
71
Protein dry weight (%)
55% - roughly 50,000 ribosomes per cell
72
True or false: Synthesis of RNA and protein is costly to cell and highly regulated
True
73
Promoter
DNA sequence that determines where transcription starts
74
Polycistronic (polygenic) mRNA
A single mRNA often codes for more than one product (in prokaryotes) -proteins translated from 1 polycistronic mRNA usually function in a related way
75
Operon
Regulatory sequences and clustered adjacent genes on DNA, transcribed into a single mRNA from a single promoter
76
True or false: Eukaryotes often make polycistronic mRNA.
False —\> they very rarely make polycistronic mRNA
77
RNA synthesis catalyzed by ________ using ___ template and \_\_\_\_
RNA Polymerase; DNA template; (r)NTP’s
78
RNA polymerase primary catalytic complex/ core enzyme:
Alpha2, Beta, Beta’, omega
79
Archaea RNA polymerase:
- much more complex than in bacteria, 11-12 subunits
80
Eukarya RNA Pol:
3 main RNA polymerases - RNA Pol II (most closely related to Archaeal enzyme) also used for making mRNA precursors
81
Bacteria RNA Pol structure looks like:
Lobster claw
82
Holoenzyme
Sigma subunit associated with the RNA Pol Core Enzyme
83
Bacterial RNA Pol Holoenzyme consists of:
Sigma subunit and core enzyme - both are necessary and active during transcription initiation
84
Function of sigma subunit
It is part of active holoenzyme; ensures that bacterial RNA polymerase binds in a stable manner to DNA only at promoter sequences
85
Are sigma subunits found in Archaea?
No - also not found in eukarya. Only found in bacteria
86
How many distinct states does RNA Pol exist in the bacterial cell?
2 distinct states: core enzyme (without sigma subunit) and holoenzyme (with sigma subunit)
87
RNA Pol contacts about ____ bp of ___ simultaneously.
50; DNA
88
RNA Pol ________ initially binds ______ to DNA
Holoenzyme; loosely
89
Why does RNA Pol initially bind loosely?
This is to search for specific sequence that it wants to find, which is the promoter sequence.
90
What happens when RNA Pol finds the promoter sequence?
The sigma subunit binds to the -35 to -10 promoter sequence; binds more tightly (different mode of binding)
91
Binding to the promoter sequence forms the \_\_\_\_\_\_\_\_
Closed complex
92
Where is the -35 to -10 region located?
On the dsDNA adjacent to where transcription will initiate
93
Open complex:
Once RNA Pol binds to promoter, there is a conformational shift within the core enzyme that creates a single stranded bubble within RNA Pol complex
94
True or false: RNA Pol can unwind DNA on its own, whereas DNA Pol requires additional enzymes to unwind the DNA as it cannot unwind DNA on its own.
True! DNA Pol III requires helicase/gyrase to unwind DNA at replication fork, RNA Pol can unwind on its own!
95
How many bp of dsDNA can holoenzyme unwind at a time?
About 10-17 bp
96
True or false: RNA Pol in closed complex can bind and join first 2 NTP’s without a primer to initiate and continue transcription.
FALSE - requires OPEN complex
97
True or false: Sigma subunit must be part of holoenzyme the entire time during transcription.
False; sigma subunit is released after about 9-10 NTP’s are polymerized. This allows RNA Pol to move away from promoter site and continue transcription in 5’ —\> 3’ direction
98
What happens after sigma subunit is released from holoenzyme?
Can be reused and bind to another core enzyme in RNA Polymerase
99
Sequence elements of bacterial promoters:
6 bp sequence centered 35 bp upstream of RNA start site (-35 sequence) and 6bp sequence 10 bp upstream (-10 sequence)
100
What is characteristic of -35 and -10 sites?
Allows for optimal spacing for sigma factor to bind while RNA Pol holoenzyme is locating promoter site
101
Is the sequence between -35 and -10 sites conserved?
No; the sequence is variable and often does not matter what the sequence is exactly. The spacing is more important.
102
True or false: Different holoenzymes recognize different promoter sequences.
True; this mainly has to do with the sigma factor that associates with the core enzyme that can recognize different promoters. Ecoli has 7 different sigma factors. Most common one is sigma 70
103
True or false: promoters are single stranded regions of DNA.
False: promoters are double stranded regions of DNA.
104
Comparing various strong promoters, sequence analysis provides \_\_\_\_\_\_\_\_\_.
Consensus sequence
105
Sigma 70 significance
Most common subunit within holoenzyme in E. coli. Used for expression of a lot of housekeeping genes, even in bacteria that have multiple forms of sigma subunit.
106
-35 region that sigma 70 recognizes:
TTGACAT —\> with TTG most important for binding
107
-10 region that sigma 70 recognizes:
TATAAT —\> 2nd A and last T are important for binding
108
True or false: Changes in highlighted bases within the -35 and -10 regions weaken the promoter significantly.
True; this can vary transcription over 100x with these changes alone
109
Sigma 4 (within sigma subunit) recognizes \_\_\_\_
-35 site
110
Sigma 2 (within sigma subunit) recognizes\_\_\_
-10 site
111
What is meant by short RNA-DNA in heteroduplex?
There is always a short region of RNA associated with DNA right near the catalytic center of RNA synthesis within bubble of open complex.
112
RNA Pol has __________ in its progression down a DNA molecule during transcription.
Periodic pauses; some are random, some at pause sequences.
113
Which end of RNA is outside of RNA pol, in the cytoplasm?
5’ end
114
True or false: termination relies on specific sequences in DNA.
False; relies on specific sequences in RNA; it is how the sequences act in the RNA molecule that impact the termination process.
115
What are the two main types of termination?
Rho dependent and Rho independent
116
What kind of processing happens in bacterial RNA’s?
rRNA and tRNA are processed from larger transcripts and are then folded into stable, functional forms
117
What do rRNA operon consist of?
A few genes encoding for different rRNA subunits, such as a gee for 16S rRNA and 23S rRNA. Sometimes there are genes for tRNA’s in the middle.
118
True or false: All bacterial rRNA’s transcribed from operon with this basic structure, but the number and specific tRNA genes will vary.
True
119
What are the 3 rRNA species in bacteria?
16S, 23S, 5S
120
What is significant about E. Coli 16S rRNA?
It binds many ribosomal proteins at specific locations during assembly of ribosomes. Over 50 nucleotides long.
121
Why does the 3’ end of 16S rRNA remain open?
It is essential in identifying a portion o the mRNA to identify the start codon for protein synthesis (translation) to be initiated.
122
What is the 30S aka small subunit comprised of?
Just the 16S rRNA plus 21 proteins make up the small subunit
123
What is the 50S subunit comprised of?
5S and 23S rRNA plus 34 proteins; makes up the large subunit 23S is the conserved catalytic site needed for peptide bond formation.
124
True or false: ribosomal RNA is very unstable and has a very short half life.
False: rRNA’s in ribosomes are very stable and their half lives can last hours.
125
What happens during tRNA maturation?
Once the initial primary transcript is established, it will be trimmed down and many bases become modified to allow specificity during aa attachment
126
Characteristics of tRNA’s
- Each different tRNA is coded by a different gene, even though there are several conserved elements.
- 73-93 nt long
- Anticodon loop is ssRNA, which will bp with codon on mRNA
- Same CCA sequence at the 3 prime end where AA is attached to OH in ribose
127
What does aminoacyl tRNA synthetase do?
Attaches amino acid to 3’ end of tRNA molecule
1. Attaches ATP to carboxyl end off AA making AA-Amp
2. Transfers AA to 3' OH on 3' end of tRNA releasing AMP
128
How are rRNA genes transcribed?
They are found within a single operon; usually transcribing a 16S, 23S, and 5S and potentially a tRNA genes in the middle.
The number of subunits and specific tRNA genes vary.
129
Where is the acceptor end of the tRNA found?
3’ end
130
Where is the anticodon found on the tRNA?
Anticodon loop, opposite from the acceptor end
131
True or false: anticodon loop is dsDNA
False; it is ssRNA in order to bp with codon on mRNA
132
What is one of the most common amino acids used during protein synthesis?
Leucine recognized by 6 different tRNA codons
133
What is wobble base pairing?
Flexible base pairing between he mRNA codon and the anticodon on the tRNA at the 3rd position in the codon (1st position in the anticodon)
134
Why does wobble base pairing work?
- some tRNA’s can have an Inosine base (I) at the 1st position
- there can also be a modified base in a base adjacent to the anticodon that changes the shape of the anticodon to allow for more flexible base pairing
Ala specified by one tRNA that recognizes GCX codons
135
What is accomplished via wobble base pairing?
Allows some amino acids to have several codons recognized by 1 tRNA.
136
True or false: Bacterial and Archaeal mRNA’s often have multiple RBS within polycistronic mRNA to allow translation of all genes.
True
137
How is the peptide released during translation termination?
1. Stop codon reached- can't bind any tRNA
2. Release factors bind to the empty A site
3. A conformational shift at the P site cleaves the carboxy-terminus of the polypeptide from the 3’ end of the tRNA which releases the polypeptide.
138
What is the translation termination complex?
The ribosome is no longer associated with the polypeptide but has not dissociated from its subunits yet.
There is an empty tRNA in the P site of the ribosome and
Peptide release factors are still bound in the A site and Ribosome recycling factors and IF3 cause 70S ribosome to release mRNA and disassociate into 30S and 50S subunits.
139
What is different about archaea and eukarya transcription initiation?
Uses TATA Binding Proteins (TBP) and other transcription factors (TF) that binds to promoter site on DNA first which then allows RNA polymerase to bind
140
How stable is the DNA polymerase transcription complex?
Very! It can kick other proteins that are in their way on the DNA off.
141
How does Rho-independent transcription work?
While the transcriptional complex is paused
1. Two GC rich regions near the 3' end base of mRNA base pair forming a hairpin loop.
2. There is a sequence of mRNA UUU's paired to AAAs on DNA at 3' end
3. Stable bonds in hairpin stress and weaken U- A bonds (sometimes NusA stabilizes hairpin more)
4. Complex falls apart
142
Rho dependant termination steps?
1. Rho binds to C rich site on mRNA
2. Translocates 5' ---\> 3' up the RNA toward RNA poly DNA complex
3. When Rho reaches a paused complex it unwinds complex and terminates transcription
143
Translation elongation steps?
1. tRNA in P site with attached AA with free carboxyl end
2. EF-Tu-GTP bind tRNA with next AA and guides to A site
3. GTPis hydrolyzed releasing EF-Tu-GTP (it is restored to GTP after leaving)
4. Peptidyltransferase activity catalyzes new peptide bond from carboxyl end of existing AA to the amino end of incoming AA transferring the peptide chain to tRNA in A site
5. Transferase EF-G-GTP with GTP bound comes in hydrolyzes GTP to GDP and ratchets tRNA in P site to E site and tRNA from A site moves to P site
6. When new tRNA is bound to A site tRNA in E site is released (only 2 tRNA bound at one time)
144
How many GTP does it take to attach one AA to an existing chain?
2 GTP
145
What is the rate of translation?
15-16 peptide bonds per second
146
What comprises the bacterial initiation complex and how does it recognize the start codon?
30S ribosomal subunit with IF 3 bound in E site, IF 1 binds in A site
This binds to mRNA and 30S complex identifies the start codon by using the open 3' end of 16S subunit to bind to 3' end of the Shine Delgarno sequence or RBS on 5' side of the start codon
147
Once the start codon is recognized by a small 30S subunit what happens?
IF-2 binds fMet-tRNA which then the 50S large subunit comes in and binds to fMet-tRNA positioning it in P site.
First EF-Tu-GTP bound tRNA comes in and binds at A site to make first peptide bond.
148
What makes up the RBS or Shine Dalgarno Sequence and where is it located on mRNA?
Purine rich sequences about 5-10 bases on 5' site of the start sequence
149
What are start sequence codes?
90% AUG
9% GUG
1% UUG
0.1% CUG
150
True or False- All start codons in bacteria bind fMet-tRNA.
True
Other than start codons AUG later in mRNA bind Met-tRNA
151
What does archaea bind first in translation?
Normal Met-tRNA
152
Examples of bacteria that target cell wall synthesis?
Penicillins, Cephalosporins, Bacitracin, Vancomycin, Monobactams
153
Antibiotics that inhibit DNA replication?
Quinolones, Ciprofloxacin
154
Antibiotics that inhibit RNA polymerase?
Rifampin, Pyronins
155
Antibiotics that inhibit protein synthesis?
50S subunit- Macrolides aka erythromycin
30S subunit- Aminoglycosides aka Gentamycin
Tetracyclines aka doxycycline
156
