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nucleic acids > midterm 2 > Flashcards

midterm 2 Flashcards

1
Q

transcription and replication is chemically and enzymatically very similar to replication, how is it different?

A
  • the new strand is made from ribonucleotides, not deoxyribonucleotides
  • has RNA polymerase that catalyzes RNA synthesis without the need for a primer
  • the RNA product does not remain base-paired to the template DNA strand but is displaced only a few nucleotides behind where each new ribonucleotide is added (therefore, multiple RNA polymerases can transcribe the same genes at the same time, each following closely behind another-allows large number of transcripts of a single gene in a short time)
  • less accurate than replication (less proof reading mechanisms)
  • copies only a region and makes many copies vs the entire genome and only once per cell cycle
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2
Q

describe the structure of RNA Pol

A

because RNA pol performs essentially the same reaction in all cells from bacteria to humans, the structure of RNA Pol of these organisms share many features

  • > mostly all are made up of multiple subunits that perform the same task (some phage, organelle RNA Pols are single subunit)
  • > shape of each enzyme resembles a crab claw:
  • the two pincers are predominantly the the two largest subunits of each enzyme(B’ and B for bacterial cases and RPB1 and RPB2 for Euk)
  • the active site is at the base of these pincers in regions called “active centre cleft” and contains one tightly bound Mg2+ ion
  • the active site has many channels to allow rna, dna and ribonucleotides into/out of active centre cleft
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3
Q

compare types of eukaryote RNA Pol and bacteria

A

bacteria have only a single RNA polymerase :
1. RNA Polymerase core enzyme - capable of synthesizing RNA
eukaryotes have 3:
1. Pol 1 - transcribing specialized, RNA encoding genes (the large rRNA precursor gene)
2. Pol II -transcribes most genes (all protein coding genes)
3. Pol III -transcribing specialized, RNA encoding genes (tRNA genes, some small nuclear RNA genes and the 5S rRNA gene)
* Pol IV and Pol V are found only in plants where they transcribe small interfering RNAs involved in transcriptional splicing (both similar to Pol II)

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4
Q

describe the transcription step of initiation

A

a promotor= DNA sequence that initially binds the RNA polymerase (with initiation factors) and once promotor-polymerase complex formed it will undergo structural changes required for initiation to proceed

  • DNA unwinds where transcription will start producing a transcription bubble of single stranded DNA
  • Transcription occurs in 5’->3’ direction and ribonucleotide added to the 3’ end of the growing chain (only one strand acts as the template unlike in replication)
  • promotors determine which DNA stretch is transcribed and is the most common step at which regulation is imposed
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5
Q

describe the transcription step of elongation

A

once RNA Pol has made a short stretch of RNA (~10 bases), it shifts into elongation phase

  • RNA Pol further undergoes con. change to tightly bind template more firmly
  • the enzyme unwinds the DNA in front and rewinds it behind, it dissociates the growing RNA chain from the template as it moves along AND it performs proofreading functions (All functions from one enzyme)
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6
Q

describe the transcription step of termination

A

once RNA polymerase has transcribed the length of the gene(s), it must stop transcribing and release the RNA product (as well as dissociating from the DNA itself)

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7
Q

describe the three defined steps of transcription initiation

A
  1. initial binding of polymerase to a promotor to form a closed complex (DNA remains double stranded and enzyme bound to one face of helix)
  2. closed complex undergoes transition to open complex where the DNA strands separates ~14 base pairs around the start site to form bubble exposing template strand and allows first 2 RNA bases to be joined. “initial transcribing complex” adds first 10 bases inefficiently and decision is made to either abort or elongate. the enzyme often releases short transcripts (each less than 10 nucleotides) and then begins synthesis again
  3. promotor escape-
    once an enzyme makes a transcript longer than 10 nucleotides, it is said to have escaped the promotor and has formed a stable ternary complex, containing enzyme, DNA and RNA. this is the transition to the elongation phase
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8
Q

what is the bacterial RNA polymerase holoenzyme ?

A

it is the form of enzyme transformed from the bacterial core RNA Polymerase that only initiates transcription at a promotor site because an initiation factor (called a sigma protein) was added to it
-the dominant sigma factor in E coli is Sigma^70

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9
Q

describe the structure of a promotor that is recognized by a RNA polymerase enzyme in which sigma^70 is added to it

A

the promotor has two conserved sequences each 6 nucleotides long and separated by a nonspecific stretch of 17-19 nucleotides

  • the two defined sequences are centred at ~10bp and ~35bp upstream the site where RNA synthesis starts
  • the two sequences are thus called -10 and -35 regions or elements (these sequences are not identical for all sigma^70 promotors)
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10
Q

what are consensus sequences?

A

they are sequences derived from comparing many different promotors and reflects preferred -10 and -35 regions, separated by the optimal spacing of 17 bp
(few promotors have this exact sequence but most differ from it by only a few nucleotides)

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11
Q

what is meant by the strength of a promotor and how is its strength influenced

A

promotors with sequences closer to the consensus are generally “stronger” than those that match less well

  • strength= how many transcripts it initiates in a given time
  • strength is influenced by how well the promotor binds polymerase initially, how efficiently it supports isomerization and how readily the polymerase escapes the promotor
  • this explains why promotors are so heterogeneous: some genes need to be expressed more highly than others
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12
Q

what is an UP-element

A

it is an additional DNA element that binds RNA polymerase and is found in some strong promotors
-it increases Pol binding by providing an additional specific interaction between the enzyme and the DNA

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13
Q

what is meant by an extended -10 element

A

Some promotors lack a -35 region and instead have an extended -10 element which comprises of the standard -10 region with an additional short sequence at its upstream end
-extra contacts made between polymerase and this extended region compensate for the missing -35 region

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14
Q

what is a discriminator

A

found just downstream the -10 element and binds polymerase. the strength of this interaction influences the stability of the complex between the enzyme and promotor

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15
Q

describe the 4 regions of the sigma^70 factor and how they bind to the promotor sequence

A

sigma 1: recognizes the discriminator by an alpha helix
sigma 2: recognizes -10 element and has helix that has amino acids that interact with bases non template strand to stabilize melted DNA
sigma 3: recognizes the extended -10 element
sigma 4: recognizes -35 element, has two helices that form a DNA binding motif (helix-turn-helix) where one helix inserts into major groove and interacts with the bases in the -35 region and the other lies across the top of the groove contacting the DNA backbone

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16
Q

how is the UP-element of a promotor sequence recognized by a holoenzyme?

A

unlike the other elements within the promotor, the UP-element is not recognized by sigma but instead recognized by the carboxy-terminal domain of the alpha subunit, called ⍺CTD which is connected to the ⍺NTD (by a flexible linker) which is embedded in the body of the enzyme

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17
Q

describe the melting of the -10 elements by σ region 2

A

melting is when RNA Pol transitions from closed to open complex and the DNA double strands open to reveal the template and nontemplate strands (melting occurs between positions -11 and +2)
in bacteria, this transition is called isomerization and it is a spontaneous conformational change not requiring ATP
->two bases in the non-template strand of the -10 element flip out and insert into pockets of RNA Pol within the σ protein (these interactions stabilize the single stranded form of the -10 element and derive melting of the promotor region)
-isomerization is irreversible and usually means transcription will initiate (in contrast, closed complex formation is readily reversible)

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18
Q

describe the 5 channels of RNA Polymerase

A

the rNTP-uptake channel allows ribonucleotides to enter the active centre
the RNA exit channel allows the growing RNA chain to leave the enzyme as it is synthesized during elongation
the remaining three channels allow DNA to enter and exit from the enzyme:
the downstream DNA channel(between the pincers) allows downstream/yet to be transcribed ddDNA to enter the active centre cleft
the non-template strand exits the active centre cleft through the non-template-strand (NT) channel and travels across the surface of the enzyme
the template strand follows path through the active centre cleft and exits through the template-strand (T) channel
*the double helix reforms at -11 in the upstream DNA behind the Pol enzyme

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19
Q

describe the role of the σ subunits 1.1 region during Pol closed and open complex formation

A

acts as a molecular mimic:

blocks active site in closed conformation and moves out of the way during open complex formation

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20
Q

why is it such an impressive feat that RNA Pol can initiate a new RNA chain on a DNA template without a primer

A

because it requires DNA template strand be brought into Pol active site and held stably in a helical conformation and the initiating ribonucleotide be brought into the active site and be held stably on the template while the next NTA is presented with correct geometry for the polymerization to occur.

  • > this is difficult partly because RNA Pol starts most transcripts with only an A which bind the T on the template strand with only 2 H bonds
  • initiating rNTP is held tightly in correct orientation by extensive interaction of the holoenzymes σ subunit
  • this RNA Pol initiating process requires very specific interactions
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21
Q

describe the three general models of how RNA Pol active site translocates along the DNA template during initial abortive cycles of transcription where the enzyme makes short transcripts of ~10 bases
-which model is believed to be correct?

A
  1. transient excursion:
    brief cycles of forward and reverse translocation of RNA Pol. the enzyme leaves the promotor and translocates a short way along template making a short transcript and then aborts and releases transcript and returns to original position on promotor
  2. inchworming:
    RNA Pol active site extended over template, making a short transcript before aborting, retracting active
    site to body of the enzyme still at the promotor & restarting synthesis of RNA
  3. scrunching:
    RNA Pol scrunches/pulls in and unwinds downstream DNA into stationary RNA Pol enzyme complex, causing single-stranded DNA bulges in the active site
    *this model is said to be correct based on experiments
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22
Q

describe how promotor escape happens

A

=All promoter-RNA Pol and RNA Pol core-δ subunit interactions need to be broken for RNA Pol to escape promoter region
once transcript is longer than 10 bases, it can no longer fit in the enzyme and starts to go through the RNA exit channel. however, there is a region of the σ factor (region 3/4 linker) that acts as a molecular mimic, mimicking RNA. this σ region lies in the middle of the RNA exit channel and must be ejected in order for the growing RNA strand to be elongated out of the enzyme (this process can take the enzyme several attempts and hence the abortive transcripts)
-the molecular mimic may be lost once it is displaced
-then the scrunched DNA is released and rewound. the concomitant collapse of the transcription bubble may be what provides the energy required for RNA Pol to break free from the promotor and dislodge the σ factor from the core

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23
Q

how does the elongation polymerase synthesize RNA

A

only ~9 bp of rNTPs of the growing RNA chain paired to DNA template at any given time; the remainder of the RNA chain is peeled off and directed out of the enzyme through the RNA exit channel
-the size of the DNA bubble remains constant throughout elongation because as 1 bp is separated ahead of the processing enzyme, 1 bp is formed behind it

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24
Q

describe the two proofreading functions of RNA Polymerase

A
  1. pyrophosphorolytic editing:
    the enzyme uses its active site in a simple back reaction where it removes the incorrect ribonucleotide by reincorporation of pyrophosphate (PPi) and adds the correct rNTP
  2. hydrolytic editing:
    the polymerase back-tracts by one or two nucleotides and cleaves off the RNA product, removing the error-containing sequence (this type of editing is stimulated by Gre factors (the elongation factor) which enhance this type of editing but also serve as elongation stimulating factors (makes sure Pol elongates efficiently and overcomes “arrest”)
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25
Q

what happens If RNA Pol encounters e.g., DNA lesion ?

A

it cannot proceed and needs to be pried off to allow for DNA repair
-the cell has machinery to remove the arrested Pol and recruit repair enzymes (in particular the endonuclease, UvrABC), the repair that follows is called transcription coupled repair and is done by a single protein called TRCF(Transcription Repair Coupling Factor) which is an excision repair protein that acts like bulldozer powered by ATP ->scans along DNA and either bumps RNA Pol to restart it or usually just displaces RNA Pol from the template strand and terminates transcription for the enzyme, but it makes way for repair enzymes and for another RNA polymerase

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26
Q

what is a terminattor

A

a sequence triggering the elongation polymerase to dissociate from DNA and release mRNA. there are two types of terminators in bacteria

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27
Q

describe the Rho-dependent terminator in bacteria

A
  • requires a protein called Rho to induce termination
  • this terminator has ill-defined RNA elements called a rut site. these sites need Rho factor for them to work which is a single stranded ringed shaped protein with 6 identical subunits. Rho binds to single stranded RNA as it exits the polymerase and uses the energy from ATP hydrolysis to induce termination by pulling the RNA out of the polymerase and causes the RNA Pol enzyme to fall off
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28
Q

describe the Rho-independent terminator in bacteria

A

also called intrinsic terminators because they need no other factors to work and they consist of two sequence elements: a short inverted repeat ~20 NTPs followed by a stretch of ~8 A:T base pairs (these elements don’t effect polymerase until they’ve been transcribed (they function in the RNA rather than the DNA)

  • once the inverted repeat sequence gets transcribed, it forms a stem loop structure (hairpin) due to Self-complementary sequences, which disrupts the elongation complex and causes termination of the elongating complex either by forcing open the RNA exit channel in polymerase, or, by disrupting RNA-template interaction
  • the hairpin is only an efficient terminator when it is followed by a stretch of A:U base pairs. this is because when the hairpin forms, the growing chain will be held to the active site only by A:U base pairs and these are the weakest of all base pairs
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29
Q

what are GTFs?

A

general transcription factors

  • they are several initiation factors that are required for efficient and promotor specific initiation in eukaryotes
  • (function of many GTFs= 1 σ factor)
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30
Q

are GTFs alone sufficient to bind promotor sequences and elicit significant transcription

A

no, additional factors are required due to the nucleosomes that the DNA is incorporated into, such as DNA binding regulatory proteins, mediator complex and often chromatin modifying enzymes

31
Q

what are the four different sequence elements that make up the core promotor

A

TFIIB Recognition Element (BRE), TATA box, Initiator element (Inr) and downstream Promotor Elements (DPE, DCE and MTE)

  • > Core promoter has subsets of elements, not necessarily all of them
  • > Inr element is the most common
32
Q

what are regulatory sequences?

A

Sequence elements that are required for accurate/efficient transcription in addition to the core promotor sequence elements

  • these are typically upstream the core promotor and are grouped into various categories based on location, the organism and their function
  • all of these DNA elements bind regulatory proteins (either activators or repressors) which help or hinder transcription from the core promotor
  • can be located far from the promotor on which they act
    ex. enhancers and silencers
33
Q

what is a Pre-initiation complex

A

the complete set of general transcription factors and polymerase II bound together at the promotor and poised for initiation is called the preinitiation complex
Pre-initiation complex= GTFs + RNA Pol + promoter
-this complex formation begins at the TATA element which is recognized by TFIID. The TATA region becomes distorted which allows recruitment of other GTFs and polymerase itself

34
Q

describe the order of protein transcription factor recruitment at the promotor once the TATA element becomes distorted due to TFIIB binding

A
  1. TFIIA binds & stabilizes TFIID binding
  2. TFIIB recruited
  3. TFIIF along with RNA polymerase II
  4. TFIIE
  5. TFIIH
35
Q

what mediated promotor melting and what else is required

A

Promoter melting is mediated by the helicase TFIIH and requires ATP

36
Q

what are the two steps involved in eukaryote promotor escape that was not seen in prokaryotes?

A
  1. ATP hydrolysis (besides that used for DNA melting)
  2. phosphorylation of the polymerase carboxy-terminal domain (CTD) or “tail” (addition of the phosphates help Pol shed most of the GTFs needed for initiation)
37
Q

describe the C-terminal domain “tail” of the eukaryotic RNA polymerase II

A

The tail contains a series of repeats that have a heptapeptide sequence. The number of repeats of this sequence is species-variable (# of repeats seems to correlate with genome complexity)

  • each repeat contains sites (amino acids) that may be phosphorylated by specific kinases
  • RNA Pol II tail is not phosphorylated when initially binding to promoter
  • Modification by these protein kinases controls later steps (elongation, RNA processing), can be reversed by protein phosphatases as they remove the phosphates
38
Q

describe how TATA-binding protein (TBP) binds and distorts DNA

A

TBP uses beta-sheet to recognize the minor groove of the TATA element which is unusual because normally proteins use α-helix to insert into major groove (but it is done to distort the DNA structure)

  • β sheet selects TATA element by ability to distort, not by sequence info and It favors the TATA element because the A-T bases are readily distorted
  • Phenylalanines anchor TBP to DNA and derive the DNA bending
39
Q

mediator complex is a transcriptional regulatory protein needed for DNA bound within nucleosomes, what is its role?

A

Mediator complex (associated with RNA Pol C.T.D. tail, helps recruit RNA Pol with help of activators, governs CTD kinase activity of TFIIH)

40
Q

describe the structure of the mediator compex

A

More than 20 subunits with 7 subunits showing significant
sequence homology between yeast and human
-Just 1 subunit (Srb4) essential for transcription of essentially all protein-coding genes in vivo
-If some component genes deleted from mediator complex, often leads to loss of expression of only a small subset of genes
-Organized in modules (may be variable)

41
Q

what are elongation factors and how do they bind

A

=factors that stimulate elongation(ex. TFIIS and hSPT5)

  • phosphorylation of the Pol CTD tail leads to an exchange of initiation factors for those factors required for elongation and RNA processing
  • RNA Pol II sheds GTFs & Mediator when it escapes promote and recruits elongation factors (Phosphorylation causes this switch)
42
Q

how do Elongation factor proteins (TFIIS and ELL) accelerate RNA growth ?

A

TFIIS, ELL shorten pauses of RNA Pol II during transcription
-TFIIS can also serve as RNase to remove incorrectly added RNA bases (contributes to proofreading and this helps prevent pausing due to errors)

43
Q

compare proofreading abilities of TFIIS (euk) and GreB (prok)

A

both stimulate an inherent RNase activity in the polymerase that is not part of the active site of the enzyme

  • > this helps prevent pausing due to errors
  • > TFIIS and GreB appear to act in analogous ways although structurally unrelated -this is the so-called “local editing function”
44
Q

how does RNA Pol transcribe in the presence of histone proteins?

A

Elongation in eukaryotes means histones packing the DNA must be displaced and a protein called FACT (FAcilitates Chromatin Transcription) is needed to cleat the histones

45
Q

describe FACT structure/function

A

FACT made up of 2 proteins that bind to and disassemble histone dimers ahead of RNA Pol II
-FACT subunits are specific for the two types of histone heterodimer
-After DNA transcription, FACT will then reassemble the
histone dimers again behind the RNA Pol enzyme

46
Q

what are the 4 RNA processing events that need to occur before the RNA is exported from the nucleus

A

1) Capping of the 5’ end
2) Splicing (most complicated! Removes introns – noncoding mRNA)
3) Polyadenylation of the 3’ end
4) RNA editing

  • > Proteins involved in elongation also involved in RNA processing
  • > Elongation factor recruits, then activates the RNA capping enzyme and another elongation factor recruits RNA splicing enzymes to RNA Pol
47
Q

what is capping

A

it is the first RNA processing event that involves the addition of a modified methylated guanine base to the 5’ end of the RNA transcript by an unusual 5’ - 5’ linkage involving three phosphates

48
Q

describe the 3 enzymatic steps for the creation of the 5’ cap

A
  1. RNA triphosphatase:
    a phosphate group is removed from the 5’ end of the transcript (the transcribed pre-mRNA)
  2. Guanylyl transferase:
    - > catalyzes the condensation of GTP with the 5’ end of the pre-mRNA and Pyrophosphate is released (GMP added)
  3. Guanine-7-methyl transferase:
    - >methylates the terminal guanosine nucleotide using S-adenosylmethionine (AdoMet) as the required cofactor
49
Q

what is the purpose of the “cap” or methylated guanine added to transcript

A
  • Protects mRNA from degradation
  • Increases translational efficiency
  • Transports mRNA to cytoplasm
  • Promotes splicing of first intron
  • 5′ RNA cap formation begins when message is only 20-40 bases long-> early!
  • 5′ cap helps to recruit ribosome to mRNA to start translation; after capping, CTD of RNA Pol is dephosphorylated and capping enzymes lost
50
Q

describe the process of polyadenylation and the 4 event due to the transfer of the polyadenylation enzymes to the 3’ end of Eukaryotic mRNA

A

RNA Pol’s CTD tail recruits enzymes needed for polyadenylylation

  • once Pol has reached the end of a gene, it encounters specific sequences that after being transcribed into RNA, trigger the transfer of polyadenylation enzymes to that RNA (these sequences are called poly-A signals), leading to 4 events:
    1. Cleavage of the message (exonuclease)
    2. Further addition of poly-A (many adenine residues) to its 3′ end mediated by poly-A polymerase and ATP
    3. RNA ‘leftovers’ in RNA Pol destroyed (RNase)
    4. Termination of transcription
51
Q

what are the two models of transcription termination after polyadenylation

A

1) “torpedo”
- >involves an RNase that recognizes the uncapped guanines in the newly synthesized, post poly A, RNA and degrades that RNA

2) allosteric
-> a critical conformational change in RNA Pol occurs
(perhaps when RNA Pol loses enzyme or interacts with other proteins)

52
Q

describe Transcription by RNA Polymerase I & III

A

RNA Pol I and III recognize distinct promoters and transcribe distinct genes(encode specialized RNA rather than protein), using distinct sets of transcription factors, but still require TBP (TATA Binding Protein, a general GTF)

  1. RNA Pol I: only transcribes ribosomal RNA precursor-encoding gene (many copies of this gene in each cell). promotor has two parts-> core element and upstream control element (UCE)
  2. RNA Pol III: transcribes transfer RNA genes, 5S rRNA genes, etc.
    - >vast majority of Pol III have weird feature where promoter is downstream Pol III transcription start site (in coding region of gene)
    - >Specialized TFIII factors required depend on type of gene being transcribed
53
Q

how can bacteria small RNAs control transcription?

A

6S RNA of E coli can bind to sigma 70 subunit of RNA Pol and down regulates transcription for many sigma 70 promotors which causes RNA Pol to shift to sigma S promotors instead

  • 6S RNA builds up in growing E. coli cells as they enter stationary phase where nutrients become depleted and cell division then stops (stress!)
  • Stationary phase causes alternative σS factor to be made, outcompetes σ70 subunit, and σS factor promotes transcription of survival proteins
  • therefore, sRNAs can serve to repress levels of one protein and promote another
54
Q

how are Small RNAs made and what is their function?

A 
Small RNAs (sRNAs, 80-110 nucleotides) made from tiny genes
-> bind to complementary target mRNAs to form double-stranded RNAs
->sRNAs therefore cause mRNAs destruction by RNase, blocking translation
(but sRNAs can in some cases stimulate translation…)
55
Q

what is Hfq

A

Host Factor Q protein is a bacterial RNA chaperone that helps sRNA bind mRNA target
->Hfq facilitates binding between short, imperfect matching of mRNA-sRNA and then stabilizes it (not very stable since not many base pairs…)

56
Q

how is σS factor (S=Stationary phase) translation affected by mRNA-sRNA binding

A

either stimulates or inhibits translation of σS factor
Translation activation:
->sRNA pairing unmasks the ribosome binding site by removing a RBS-blocking RNA region
or
Translation repression:
->inhibits ribosome binding by masking the RBS, RNA Pol can’t bind

57
Q

what are Small antisense RNAs

A

they are regulatory RNAs in bacteria that are encoded by the strand opposite the coding strand of a gene and act through homologous base pairing to inhibit expression of the mRNA produced from that gene
*they act only on the gene in which they are made (cis)

58
Q

what are riboswitches

A

are “built-in” metabolite sensors using RNA sequences

  • control expression of the genes within whose mRNA they reside
  • found within the 5’-untranslated regions (5’-UTRs) of genes they control
  • can regulate expression at the level of transcription or translation through changes in RNA secondary structure
  • Riboswitches usually upstream of genes involved in synthesis of metabolite that the riboswitch responds to
59
Q

what are the two components of riboswitches?

A
  1. the aptamer:
    - >70-200 nucleotides long and is the 5′ untranslated region (UTR) region that binds a metabolite
  2. expression platform:
60
Q

what happens when the aptamer of the riboswitch binds the small-molecule ligand (metabolite)

A

it undergoes conformational change which causes a change in the secondary structure of the adjoining expression platform

  • these con. changes alter expression of associated gene by either (1) terminating transcription or (2) inhibiting the initiation pf translation
  • this happens in response to the concentrations of small molecules/metabolites
61
Q

what are the two examples of SAM-sensing riboswitch (Binding of SAM causes one of two different rearrangements in aptamer and expression platform)

A
  1. regulating transcription termination (=Attenuation):
    - > blocks any further RNA Polymerase transcription of the transcript because a stem loop structure is formed when SAM binds to the amptamer which acts as a transcriptional terminator (SAM stabilizes this secondary structure)
  2. regulating translation initiation:
    - >initiation prevented by making a structure with the RBS site
    - > SAM binds to aptamer and causes the stabilization of a secondary structure (stem loop) that sequesters the RBS and blocks ribosomes from initiating translation
62
Q

Riboswitches can bind 8 recognized metabolites, each with unique structures, but what else can riboswitches respond to besides metabolites?`

A

uncharged tRNAs (through the aminoacyl tRNA synthetase gene)

  • When charged tRNA amount falls, uncharged tRNAs (only!) can bind to the riboswitch structure and cause it to flip from “off” to “on” (forms anti-termination structure)
  • charged rRNA does not fit the binding pocket provided by the leading RNA secondary structure
  • > Riboswitches can bind and thus respond to simple and complex metabolites
63
Q

What is (DNA) sequencing?

A

Determining the linear order of nucleotide bases in DNA is sequencing
-> targets genome (chromosomal DNA) and transcriptome (All RNA transcripts)

64
Q

compare genome vs transcriptome

A

genome: chromosomal DNA
->Coding sequences (genes) and Non-coding sequences (promoters, terminators, enhancers, intergenic regions)
->Sequence content is unchanging regardless of cell condition
transcriptome: All RNA transcripts
->Messenger RNA (mRNA), Ribosomal RNA (rRNA)
Transfer RNA (tRNA), Non-coding RNA (miRNA, etc)
->Sequence content is variable
-A snapshot of which genes are expressed upon harvesting RNA
-RNA converted into DNA, then sequenced

65
Q

what are the First generation sequencing methods?

A
  1. Maxam-Gilbert (obsolete, 1973)

2. Sanger didexoynucleotide with radiolabels (1977)

66
Q

what are the Next generation sequencing methods?

A 
Roche 454 (2005-2016)
Ion torrent (2011)
Pacific Biosciences (2011)
Illumina (2011)
Nanopore (2012…)
*Next-generation sequencing technologies have surpassed Sanger sequencing
67
Q

what are the basis of Sanger-type sequencing

A

based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
(Sanger sequencing uses terminator dideoxyNTP(with base-linked fluorophore) in place of deoxyNTPs by removing an oxygen atom from the ribonucleotide)
steps:
-primer added, DNA Pol adds normal dNTPs using complementary base pairing of template strand but polymerase enzyme can no longer add normal nucleotides onto the DNA chain once the ddNTP is added.
-The extension has stopped and now need to identify what the sequence is. We identify the chain terminating nucleotide by a specific fluorescent dye, (4 specific colors). -Sanger sequencing results in the formation of extension products of various lengths terminated with dideoxynucleotides at the 3′ end.
-The extension products are then separated by Capillary Electrophoresis (and with acrylamide gels) which separate the extension products by size
-A laser excites the dye labeled DNA fragments as they pass through a tiny window at the end of the capillary (Fluorescence of each ddNTP-fluorophore detected, correlated to C, G, T, A)
-DNA sequence assembled by reading each signal

68
Q

why has sequencing moved from sanger sequencing to First generation sequencing

A
  • > Sanger-type too slow, too expensive for genomes (>1kb)
  • > Sanger-type requires knowledge of DNA sequence for primer design, NGS needed to escape this requirement
  • > Exploit recent technological advances (better detectors)
  • > Clinical-focused market will be $15 billion by 2025
  • > Has revolutionized genome, transcriptome sequencing
69
Q

describe Ion Torrent sequencing

A

DNA sample chopped, ‘sticky’ adaptors (A,B)
added to ends of DNA fragments

DNA diluted to 1 DNA molecule per bead

DNA amplified by emulsion PCR

Nanopores/wells small enough to captures only 1 bead-tethered DNA

Sequential addition of dNTPs :
dATPs, wash, dGTPs, wash, dTTPs, wash, dCTPs
->Adding dNTP releases a proton ion and an Ion detector senses released H+ during enzymatic addition of dNTP
->2 or more of same dNTP added to DNA increases signal, can cause problems

70
Q

Pacific Biosciences sequencing

A

DNA polymerase fixed to platform captures,
synthesizes 1 molecule of DNA (no PCR needed)

Uses tiny wells with excitation light shining from
below, but light won’t go far into well

Incoming nucleotide tagged with phospho-linked
fluorophore bound by DNA polymerase

Real time sequencing:
detects fluorescence of excited, tagged nucleotide
during incorporation as DNA synthesized,
signal lost after cleavage
-Different fluorophore incorporation generates transient emitted light
-makes sequential bursts of light as NTPs added which corresponds to the different nucleotides
-SMRT sequencing harnesses DNA Pol’s natural ability to synthesize ~10+ bases per second with many Pol acting at once leading to high speed and long read sequencing (DNA is sequenced as it is being replicated in real time)

71
Q

Illumina Genome sequencing basis of using stop-flow terminating fluorescent dNTPs?

A

steps:

  1. universal primers immobilized on glass surface inside flow cell
  2. genomic DNA broken up into sequencing templates with added tail that matches the primer as well as a labeled nucleotide and then ready to be loaded into flow cell
  3. DNA is immobilized to the primers within the cell
  4. illuminate glass surface with laser and use electronic camera connected to a microscope to visualize the positions of the template:primer on the surface. Now dye molecules are cleaved/washed away
  5. DNA polymerase will catalyze the addition of the fluorescently-labelled nucleotide to the appropriate primers
  6. wash out DNA Pol and unincorporated NTPs, the 3′-OH is blocked, terminating reaction
  7. record positions of incorporated nucleotides by again illuminating surface with a laser and camera
  8. remove the fluorescent label on each nucleotide
  9. Remove the 3′OH blocking molecule and flow in DNA polymerase and fluorescently-labeled nucleotides. Polymerase will catalyze the addition of the next labelled nucleotides. Repeat this as many times as you want to get the desired read length.
    * 4 nucleotides each tagged with a different fluorescent dye ->Can flow all 4 across surface simultaneously
72
Q

Nanopore sequencing basis

A

= uses DNA bases pulled thru
tiny pores, altering transmembrane currents

->tiny pores can ‘sense’ which base is being passed through it
->Nano-pores embedded in artificial membrane
->Electrical current across membrane can be
disrupted by compounds in nanopore
->Enzyme pulls DNA through 1 base at a time, giving
DNA sequence by correlating electrical current
change to corresponding base

No drop-off in sequence quality over time,
very long reads?

73
Q

what is DNA sequence fragment assembly

A

Sequencing reads are aligned and assembled into contig DNA (overlapping DNA segments that together represent a consensus region of DNA)

  • Sequence assembly is a major computational challenge for bioinformatics
  • Many ‘reads’ of DNA sequence need to be assembled into DNA sequence (coding and/or noncoding)
  • Using overlapping sequences, small fragments are assembled into a big sequences
74
Q

how does RNA sequencing (RNA-seq) compare to DNA sequencing

A

Sequencing step is the same as DNA sequencing
but preparation of the sample is different:
-RNA isolated and quality-controlled
-RNA reverse transcribed into cDNA by reverse -transcriptase, RNA destroyed
-DNA sequenced
->Better than traditional microarrays since you don’t need to know sequence, can discover sequence variants
*RNA from transcriptomes are converted into DNA, then sequenced

nucleic acids (5 decks)

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