Unit 7 Flashcards
(101 cards)
1
Q
rRNA
A
A class of RNA molecules serving as the components of ribosomes, the complexes that carry out the synthesis of proteins
2
Q
mRNA
A
A class of RNA molecules that are translated by the ribosomes to make proteins. They are intermediaries, carrying information for the synthesis of a protein from one or a few genes to the ribosome
3
Q
tRNA
A
Adaptor molecules that faithfully translate the information in mRNA into a specific sequence of amino acids
4
Q
Identify the pentose ring (ribose vs deoxyribose)
A
RNA has a -OH group at the 2’ carbon making it ribose. DNA has a -H at the 2’ carbon making it deoxyribose
5
Q
Which nitrogen bases are found in DNA and RNA?
A
DNA: ACTG
RNA: ACUG
6
Q
Distinguish between monocistronic and polycistronic mRNA?
A
If a single mRNA carries the code for only one polypeptide, the mRNA is monocistronic.
If a single mRNA carries the code for two or more different polypeptides the mRNA is polycistronic
7
Q
Is eukaryotic mRNA monocistronic or polycistronic? What about prokaryotic?
A
- Eukaryotic: monocistronic
- Prokaryotic: polycistronic
8
Q
Is DNA usually double or single stranded?
A
Double stranded
9
Q
Is RNA usually double or single stranded?
A
Single stranded
10
Q
Does single stranded RNA have any double stranded regions? Point out a hiarpin loop.
A
Yes, complementary strands in two single strands of RNA (or within a single strand of RNA that folds back on itself to align the residues can pair with each other)
- Hairpin loops form between nearby self-complementary sequences (palindromic sequences)
11
Q
Within double stranded regions of RNA, define two types of base pairs
A
AU
GC
12
Q
What are features of a double stranded RNA?
A
- Bulge when there are mismatched pairs
- Internal loops (when there are also mismatched pairs)
- Hairpin when there are palindromic sequences
13
Q
Give an overview of transcription by RNA polymerase
A
- The reaction involves two Mg2+ ions coordinated to the phosphate groups of the incoming nucleoside triphosphates (NTPs) and the 3 Asp residues, which are highly conserved in the RNA Polymerases of all species. One Mg2+ facilitates the attack of the 3’-hydroxyl group on the alpha phosphate of the NTP; the other Mg2+ facilitates displacement of the pyrophosphate
- RNA polymerase and the transcription bubble move from left to right along the DNA. The DNA is unwound ahead and rewound behind as RNA is transcribed. As the DNA is rewound, the RNA-DNA hybrid is displaces and the RNA strand is extruded.
- Movement of an RNA Polymerase along DNA tends to create positive supercoils (overwound DNA causes the DNA strands to melt apart from the tension) ahead of the transcription bubble and negative supercoils (underwound
14
Q
Compare and contrast RNA polymerases and DNA polymerase I considering:
substrates
A
- RNA Polymerase: NTP
- DNA Polymerase: dNTP
15
Q
Compare and contrast RNA polymerases and DNA polymerase I considering:
Primer needed
A
- RNA Polymerase: does not need primer
- DNA Polymerase: needs a primer
16
Q
Compare and contrast RNA polymerases and DNA polymerase I considering:
Direction of Synthesis
A
Both are synthesized in the 5’ to 3’ direction
17
Q
Compare and contrast RNA polymerases and DNA polymerase I considering:
Nucleophilic attack in bond formation
A
Both have a 3’-OH group act as a nucleophile, attacking the alpha phosphate of the incoming NTP or dNTP and releasing PPi
18
Q
Compare and contrast RNA polymerases and DNA polymerase I considering:
Role of pyrophosphate and pyrophosphate in the overall reaction
A
For both, the hydrolysis of PPi drives the overall reaction towards the products (production of lengthened RNA/DNA)
19
Q
Compare and contrast RNA polymerases and DNA polymerase I considering:
Requirements for a template
A
- RNA Polymerase: Yes (DNA template)
- DNA Polymerase: Yes (DNA Template)
20
Q
What is meant by the terms “template” and “non-template” strands
A
The DNA strand that serves as the template for RNA synthesis is called the template strand. The DNA strand complementary to the template, the nontemplate strand, is identical to the RNA transcribed (with T replaced with U)
21
Q
Does the same strand of chromosome always serve as the template strand?
A
The bottom strand acts as template for these transcripts; however it is possible to have them transcribed with the top strand (only some are transcribed with the top strand). However, it’s usually the bottom strand because that is in the 3’ to 5’ direction
22
Q
Discuss the quaternary structure of RNA polymerase
A
- 6 subunits.
- The RNA polymerase looks similar to a claw, with the pinceres formed by the Beta and Beta’ subunits
- The sigma subunit (sigma70) rests on top of the RNA polymerase and threads through the RNA exit channel
23
Q
Does RNA polymerase have 3’ to 5’ exonuclease activity? Why is it unnecessary?
A
No, RNA polymerase doesn’t. This is because nearly all RNAs are eventually degraded and replaced, a mistake in an RNA molecule is less of a consequence to the cell than a mistake in the permanent information stored in DNA
24
Q
What is meant by the term consensus sequence?
A
A sequence of nucleotides or amino acids that represent the most commonly observed sequence at a specific position in a set of related DNA, RNA, or protein sequences
ex. the -10 and -35 regions
25
Define the numbering system which is used to identify regions such as the -10 and -35 regions
The DNA base pairs that correspond to the beginning of an RNA molecule are given positive numbers, and those preceding the RNA start site are given negative numbers
*this is about DNA in relation to RNA
26
What is the significance of the -35 and -10 regions
They are both consensus sequences that serve as recognition sites for RNA polymerase and help recruit the enzyme to the promoter region
* the sigma factor initially binds to the -35 and -10 region with the RNA polymerase. This positions it correctly so the RNA polymerase itself can bind to these areas
27
What is the relationship between the "strength" of a promoter and the degree of match to a consensus sequence?
The closer the match to the consensus sequence, the stronger your promoter
Remember the consensus sequence is an average or general guess of what the nucleotide sequence in that area should look like. The closer the actual promoter sequence matches the consensus sequence, the stronger the promoter tends to be
28
What is the function of the sigma subunit?
It associates with the core enzyme of RNA Polymerase and also is responsible for recognizing and binding to specific DNA sequences within the promoter region, such as the -35 and -10 regions, introducing a large bend in DNA through the process.
29
Discuss the events in transcription initiation and elongation.
1. RNA polymerase and core sigma factor subunit bind to the DNA promoter. At this point the complex is closed (meaning the DNA strands are together)
2. Transcription bubble forms opening the complex
3. Transcription is initiated (technically, at this stage the RNA polymerase is synthesized short RNA transcripts known as abortive transcripts. Once RNA polymerase has synthesized a sufficient length of RNA to stabilize its interaction with the DNA template, it can transition into elongation)
4. Promoter clearance is followed by elongation.
5. Elongation continues, sigma 70 dissociates and is replaced by NusA
6. Transcription is terminated NusA dissociates, and the RNA polymerase is recycled
30
Is transcription usually regulated at the level of initiation, elongation, or termination? Is it ever regulated at the other levels?
Regulation can occur at any step in transcription, including elongation dn termination.
However, much of the regulation is directed at the polymerase binding and transcription initiation steps. Differences in promoter sequences are just one of several levels of control. The binding of protein sequences near or distant from the promoter can also affect levels of gene expression.
31
What does a strong promoter mean?
RNA polymerase will bind tighter so the frequency and rate at which transcription is initiated at a particular promoter is increased
32
Discuss two distinguishing features of the termination signal in the mRNA in rho independent termination of transcription in prokaryotes
- The palindromic sequence can fold back on itself, forming a hairpin structure. This disrupts the interaction between RNA and DNA template with the POLYMERASE
*this would occur if your RNA transcribed two sequences that are complementary to each other
- After the hairpin structure, the DNA has an AAAAA sequence meaning the RNA sequence will be transcribed to be UUUUU. A and U have weak interactions, so the RNA and DNA would separate from each other, leading to termination
33
Describe the sequences typically found at a eukaryotic promoter
1) The TATA box is the major assembly point for the proteins of preinitiation complexes of Pol II
2) The Initiator Sequence (Inr): the DNA is unwound here and the transcription start site is usually within or very near this sequence
3) Additional sequences around the TATA box and downstream (to the right) of the Inr may be recognized one or more transcription factors
34
How do these sequences compare to those found at a prokaryotic promoter*
- Eukaryotic transcription machinery is in the nucleus (prokaryotes don't have a nucleus)
- There are 3 types of RNA Polymerase
-Some of the promoters have a TATA box
- Requires an array of other proteins at the promoter and elsewhere to begin transcription
35
What is a transcription factor?
protein factors required for transcription by RNA polymerase II in eukaryotes
36
Discuss the formation of a primary transcript and its processing to an mRNA
*newly synthesized RNA is called primary or precursor transcript
- Transcription occurs from a 5' to 3' direction and produces a primary transcript containing both exons and introns. The introns are spliced out, producing mature mRNA. The poly A tail is added to the mature mRNA after transcription and protects the mature RNA
37
During the processing of eukaryotic mRNA a "5' cap" is added. Describe and list two functions of the cap
1. Protects mRNA from ribonucleases
2. Binds to specific cap-binding complexes of protein and participates in binding of the mRNA to that ribosome to initiate translation
- The 5' cap is a residue of 7-methylguanosine linked to the 5' terminal residue of the mRNA through an unusual 5',5' triphosphate linkage
38
Distinguish between "exons" and "introns"
Noncoding tracts that break up the coding region of the transcript are called introns and the coding segments are exons.
39
Do all eukaryotic genes contain introns? Do most?
No. The vast majority of genes in vertebrates contain introns. The occurrence of introns in other eukaryotes varies.
39
Does splicing take place at the level of DNA or RNA
Level of RNA. The introns are removed from the pre-mRNA
40
How does the mechanism of splicing happen?
- There could be different possible 5' and 3' slice sites (for the introns). This can differentiate.
- One the 5' end of the intron, there is typically a GU and on the 3' end of the intron, we have an AG
- The snRNPs will recognize those sequences through complementary base pairing between their small nuclear rnas (snRNA)
- The U1 snRNP will bind the the 5' GU end and the U2 snRNP will bind the the 3' AG end
- When the U2 binds to our transcript, it activates a nearby Adenylate (A nitrogenous base)
- After U1 and U2 bind, U4-U6 and U5 join to create the spliceosome
- The intron gets looped while the A gets closer to the 5' end of the intron
- When it gets close enough, the 2'-OH on the A actually acts as a nucleophile to attack the 5' carbon of our intron ( JUST SAY THE 3' END OF THE EXON)
- We end up with a lasso structure called a lariat.
- Now, the EXON's Hydroxyl on the 3' end will act as a nucleophile to cut off the intron and we are left with a continuous intron
41
Point out features at exon-intron junctions of group II introns that provide signals for correct splicing
UG at 5' and AG at 3'
42
What is a spliceosome?
A complex of RNA 's and proteins involved in splicing of mRNA in a eukaryotic cell
43
What is an snRNP?
Small nuclear ribonucleoproteins which make up the spliceosomes and are responsible for splicing
44
How do individual snRNAs locate the consensus sequences that flank introns>
- Each snRNP contains one class of eukaryotic RNAs known as small nuclear RNA (snRNAs). Five snRNAs (U1, U2, U4, U5, U6)
- The snurps will recognize those sequences through complementary binding between their small nulcear RNAs
45
Point out the location and identity of the nucleotide that will make the initial attack on the 3' end of the exon
Adenosine within the intron
46
What end is attacked first?
3' end of the exon
47
What is the "lariat structure"
A lasso like intermediate structure that is branched. Eventually becomes the spliced intron which leaves as a loop
48
What is the function of the poly (A) tail
- Serves as a binding site for one or more specific proteins
- Has a variety of roles in controlling transcription and translation
- Protect mRNA from enzymatic destruction
49
Discuss the key steps in the addition of poly (A) tail
1. AAUAAA site (upstream of cleavage) and the less well-defined sequence rich in G and U (downstream of cleavage) mark teh site where cleavage occurs
2. The endonuclease component of a large enzyme complex (which is associated with teh CTD of RNA Polymerase II)
3. Polyadenylate polymerase comes n and using ATP, synthesizes the poly A tail
4. Poly A tail is added to the transcript
50
Is the poly A tail encoded in the gene?
No, it is added on
51
Name the enzymes involved in making the poly (A) tail
Polyadenylate Polymerase
52
Does this enzyme add residues in a template directed manner like DNA Polymerase?
It doesn't require a template but does require cleaved mRNA as a primer
53
Clearly distinguish between maturation of mRNA and RNA editing
- RNA editing is a process that adds or deletes bases in the coding regions of primary transcripts or that change the sequence (such as by enzymatic deamination of a C residue to create a U residue)
-This is different from maturation of mRNA because it cuts out noncoded regions and adds on on poly A tail instead of altering the coded region
54
Distinguish between poly A site choice and alternative splicing. Discuss the role of each of these mechanisms in generating protein diversity from a single gene
*REFER TO FIGURES
- Alternative splicing is a process in which exons are selectively spliced in alternative ways to generate different mature mRNAs
- poly A site choice is when complex transcripts can have more than one site where poly (A) tails can form. IF there are two or more sites for cleavage and polyadenylation, use of the one closest to the 5' end will remove more of the primary sequence
- Together, alternative splicing and poly (A) site choice greatly increase the variety of proteins generated from the genomes of higher eukaryotes
55
What is meant by "RNA world"?
A hypothesis that life on Earth originated with molecules like RNA capable of storing and replicating genetic information in addition to catalyzing biochemical reactions
56
What evidence is used in support of the hypothesis?
1. Prebiotic Chemistry Experiments
2. The existence of catalytic RNAs
3. The expanding catalytic repertoire of Ribozymes
4. The structure of the ribosome
5. Extant vestiges of an RNA world
6. Progress in Search for an RNA replicator
57
Thinking back to Unit 4, recount briefly three mechanisms which control the activity of enzymes in the cell
1. Allosteric Regulation
2. Covalent modification
3. Proteolytic Cleavage
58
Distinguish between constitutive and regulated gene expression
- Constitutive gene expression is expression of a gene at constant levels
- In contrast, regulated gene expressions controls when and how much gene levels are expressed.
59
Specificity factor
Alters the specificity of RNA polymerase for given promoter or set of promoters
60
Repressor
Impede access of RNA polymerase to the promoter
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Activator
Enhance the RNA polymerase-promoter interaction
62
Operator
A region of DNA that interacts with a repressor protein to control the expression of a gene or group of genes
63
Negative regulation
REFER TO FIGURES
Regulation by means of a repressor protein that blocks transcription
TWO FORMS:
- Molecular signal causes dissociation of repressor from DNA, inducing transcription
- Molecular signal causes binding of repressor to DNA, inhibiting transcription
64
Positive regulation
REFER TO FIGURES
Regulation by means of an activator, they bind to DNA and enhance the activity of RNA Polymerase at a promoter
TWO FORMS:
- Molecular signal causes dissociation of activator from DNA, inhibiting transcription
- Molecular signal causes binding of activator to DNA inducing transcription
65
Key features of an operon
- Gene cluster
- Promoter
- Regulatory sequencesA
66
Are operons found in prokaryotes or eukaryotes?
Prokaryotes
67
Do operons encode monocistronic or polycistronic mRNA?
Polycistronic
68
Write an equation representing the hydrolysis of lactose to its component monosaccharides
Lactose + H20 --> Galactose + Glucose
69
What is the name of the enzyme which catalyzes this reaction?
Beta-galactosidase
70
Draw a diagram of the lac operon and label
71
Name each of the three structural genes and describe the functions of each
- Lac Z: encodes Beta-galactosidase (lactose breakdown)
- Lac Y: encodes galactosidase permease (allows lactose to enter the cell)
- Lac A: thiogalactosidase transacetylate (takes care of harmful by products that might form with lactose)(
72
Describe how the lac operon can be negatively regulated. What protein is involved? Name the genes that encodes this protein. What molecule acts as the inducer?
The lac operon can be negatively regulated if the lac repressor is bound to the operator. This prevents transcription from occurring because there is a protein actively blocking RNA polymerase. The lac repressor is encoded by the lac I gene, which is upstream of the lac operator and promoter. The molecule that acts as the inducer is allolactose
73
How do proteins interact at specific DNA sequences?
Regulatory proteins must recognize surface features of DNA. To interact with bases in the major groove, a protein requires a relatively small structure that can stably protrude from its surface
74
What type of interaction is most likely involved?
Hydrogen bonding
75
Within the DNA double helix, where does this interaction occur?
In the major groove
76
DNA binding motifs:
- Helix-turn-helix
- Zinc fingers
- Homeodomain
- Helix-loop-helix
- Leucine zippers
77
Helix-turn-helix
A protein domain composed of two alpha helices joined by a short strand of amino acids (beta-turn) and is found in many binding proteins.
78
Zinc fingers
A small protein structural motif that is characterized by the coordination of one or more zinc ions in order to stabilize the fold
79
Homeodomain
A conserved sequence of 60 amino acids used in the binding to DNA. Usually found in transcription factors, it is used to express genes that are related, more specifically in development to make tissues associated with one another
80
Leucine zippers
- Two alpha helices form a coiled-coiled structure and interact with each other; can be the same protein or different proteins interacting
81
Helix-loop-helix
A transcription factor DNA-binding domain formed by teh dimerization of two polypeptide chains. The dimerization domains of these proteins consist of two helical regions separated by a loop
82
Catabolite repression
A regulatory mechanism by which expression of genes required for the utilization of the second source of energy is prevented by the presence of the preferred substrate
83
cAMP receptor protein role in positive regulation of the lac operon
The activator protein will bind to the enhancer region to increase the rate of teh lac operon transcription
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cAMP
cAMP is high when glucose levels are low. It will bind to CRP to help increase the rate of transcription for the lac operon
85
DO DIAGRAM ON PAGE 68
86
List 5 important features that distinguish regulation of gene expression in eukaryotes from prokaryotes
1. Access to eukaryotic promoters is restricted by the strucutre of chromatin and activation of transcription is associated with many changes in chromatin strucutre in the transcribed region
2. Positive mechanisms are more prominent
3. Regulatory mechanisms involving IncRNAs are more common in eukaryotic transcriptional regulation
4. Eukaryotic cells have larger, more complex multimeric regulatory proteins than bacteria do
5. Transcription in the eukaryotic nucleus is separated from translation in teh cytoplasm
87
Distinguish between heterochromatin and euchromatin
Heterochromatin is chromatin in a more condensed form that is transcriptionally inactive (abt 10%). The remaining, less condensed chromatin is called euchromatin
88
Discuss the structural features of active chromatin
- Position of nucleosomes are less tightly packed
- There is presence of histone variants
- There is methylation or acetylation as the covalent modifications (by Lys or Arg residues)
89
What is meant by chromatin remodeling?
Transcription-associated structural changes to the chromatin
90
What is an enhancer?
DNA sequence that facilitates the expression of a given gene; located upstream from promoter
*activators bind to the enhancer
91
Basal transcription factors
- Proteins, present in all eukaryotic cells, that bind to promoters and help initiate transcription; general transcription factors, required for every promoter
- Directly interact with DNA
92
Transcriptional activators
- proteins that speed transcription dramatically; bind to enhancers and facilitate transcription
- Activate transcription by recruiting other proteins
93
Chromatin modification and remodeling proteins
remodeling proteins:
- reposition nucleosomes (move them out of the way)
- decondense DNA
Chromatin modification :
- Act on tails of histone proteins in nucleosomes (ex. methylation)
94
Coactivators
- Mediates interactions between the transcription activators (that may be far away) and the events at teh TATA box/promoter
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Trans regulation
A molecule that is regulated by another molecule
96
Cis regulation
Same molecule regulates its own function
97
DIscuss how riboswitches can terminate transcription, block translation, or interfere with splicing
- A segment of the mRNA has a sequence so that is actually folds into a 3D form that can bind to a ligand. This 3D structure is called the aptamer
- There is a specific ligand (TPP) that will bind to the aptamer. The binding induces a conformational change for mRNA (hairpin pops up)
- The hairpin will abort transcription. The conformational change will also block the site where teh ribosome would bind for translation, therefore translation doesn't occur
- Affects splicing **
98
Gene expression is higher eukaryotes in controlled by a new mechanism of _____
micro-RNA's
99
How do miRNA's control gene expression?
By hydrolysis of the target mRNA
100
Discuss the synthesis and processing of miRNA's
- miRNAs function in silencing genes
- They are initially transcribed as a specific gene as a precursor RNA. It has internally complement sequences and forms hairpins
- Next, an endonuclease will cut all these hairpins apart and then another endonuclease will cut teh loop itself. You end up with an RNA duplex (stRNA)
- One of the strands will be degreaded, but the other strand is complementary to its target mRNA
- Depending on how well the base pairing is of the complementary systems, you get either degradation (near-perfect complementary) or translation inhibition ( (translational inhibition)
