Lecture 7 Flashcards
(98 cards)
1
Q
Messenger RNAs (mRNA)
A
encode the amino acid sequences of
all the polypeptides found in the cell
2
Q
Transfer RNAs (tRNA)
A
match specific amino acids to triplet
codons in mRNA during protein synthesis
3
Q
Ribosomal RNAs (rRNA)
A
is the RNA component of the
ribosome, interact with tRNA during translation
4
Q
Micro RNA (miRNA)
A
post-transcriptionally regulate the
expression of genes, by binding to mRNA nucleotide sequences
5
Q
Ribosymes
A
RNA can be catalytic
6
Q
RNA Polymerase
A
Elongates an RNA strand in the 5’-3’ direction, using the 3’ hydrozyl to attack the a-phosphorous atom in the incoming nucleotide and release PPi
7
Q
In Transcription: Where is the non-template strand traversing out of?
A
Active site
8
Q
In Transcription: where is the template stand running through?
A
Active Site
9
Q
Non-Template Strand
A
Coding Strand
10
Q
Coding
A
Non-Template Strand
11
Q
DNA/RNA hybrid size
A
8 bp in size
12
Q
Transcription: NTP channels
A
Sampled in the active site (need right base matching the template)
- Right bases will incorporate
- Wrong bases will have poor Kd and get kicked out of the way
13
Q
Elongation: Footprint size
A
35 bp worth of DNA
14
Q
Initiation: Footprint Size
A
100 bp- when RNA polymerase is first associated with DNA
15
Q
What is a footprint?
A
Extent of how much RNA polymerase covers on DNA
16
Q
What is a template strand?
A
DNA Strand that serves as the template for RNA synthesis
-Other strand is called CODING because it has the same sequence as the newly made RNA molecule
17
Q
Elongation occurs as a rate of…
A
50-90 bp/second
18
Q
RNA Polymerase Core Subunits
A
a,a,B,B’,w (omega), o(omega)
19
Q
RNA Polymerase: Sigma Subunit
A
- Directs enzyme to the promoter
- The chaperone-takes the polymerase to the right promoter to the the right place where it needs to be polymerized
- Different sigmas can be used to turn on different genes all within same Core! To coordinate gene expression. they recognize own promoters
20
Q
RNA Polymerase: alpha Subunit
A
(2) alphas. Functino is assembly and binding to UP elements (sequence upstream of promoter)
21
Q
RNA Polymerase: Beta Subunit
A
Main catalytic subunit
22
Q
RNA Polymerase: B’ Subunit
A
Subunit responsible for DNA-building
23
Q
RNA Polymerase: Omega (w) Subunit
A
appears to protect the polymerase from denaturationg
- Not catalystic role
- If you take away the RNA polymerase, dalls apart faster
24
Q
Does UP element have specificity?
A
YES that interact with ALPHA subunits and Kd goes down when binding is favorable
25
Does RNA polymerase have proofreading ability?
NO so it makes mistakes every 10^4-10^5 nucleotides
-mistakes in RNA synthesis are not critical because many RNA copes are made from a single gene and they are rapidly degraded/ replaced
26
RNA polymerase binds to how many nucleotides?
100- stretching from 70 bp upstream (negative numbers) of the strat site and 30 bp downstream (positive numbers)
27
Start site is the
FIRST RNA RESIDUE that is incorporated in the RNA (+1 site)
28
Important promoter regions in E.Coli
-10 and -35
29
Pribnow Box
TATA sequence is found on -10 with a second sequence on -35.
-Sigma70 subunit binds to these seuqnces
30
A subunit binds to a third AT-RIch region. Where is it?
- AT rich region (UP element) is at position -40 to -60 in high expressed genes.
- THIS ENHANCES INTERACTION AND LOWERS Kd
31
How Variability effects binding to promoter site
Kd is lowered when RNA polymerase is lowered if some tehtering onto UP element (alpha subunits)
Kd is high when platform is layed out like this and UP element is taken away
32
Will Sigma bind/land is ONE base pair is changed?
NO-it is looking for consensous sequence at -35. a single bp change in these promotor sequences can affect the rate of transcription by order of magnitude
-GOOD Because you dont always want all genes activated at the dame time
33
Transcription Initiation: closed complex
FIRST STEP Polymerase binds to the promoter and forms a closed complex
34
Transcription Initiation: STEPS
First, the polymerase binds to the
promoter and forms a closed complex.
• Then, the DNA is partially unwound near
the -10 sequence (TAT BOX) , forming the open
complex.
• Transcription is then initiated and the
complex is converted into a
conformationally distinct elongation form. (from 100bp to 35 bp)
• The complex then moves away from the
promoter, releasing the SIGMA subunit along
the way.
• Note: a double-stranded primer sequence
is not needed.
35
Control of initiation:
Most of it is controlled in initiation but can also occur during elongation/termination
36
Control of initiation: what will differences in promoter sequences do?
One point of control where it it will require several sigma proteins with different sequence binding preferences
37
Control of initiation: Transcription Factors
Accessory proteins can bind near to the promoter and
affect transcription. These proteins can either be
activators or repressors of transcription
-Help bind by reducing Kd and enhancing Transcription
38
Control of initiation: Repressor
bind to sequence exactly where RNA polymerase want sto bind and TURN off expression
39
Transcription Elongation: RNA Chain elongation occurs in what direction?
5' to 3'
40
Transcription Elongation: Supercoiling
RNA polymerase moves the transcription bubble along the DNA strand creating a...
- POSITIVE supercoiling ahead of transcription
- NEGATIVE supercoiling behind
41
Transcription Elongation: What happens if supeercoiling is too severe?
If not releives by topoisomerase, transcription can HALT
42
Transcription Elongation: how do you get polymerase to stop for a crystal structure picture?
Analog with a nucleotide that lacks the 3' hydroxyl (nucleophile) used to STOP
43
Transcription Elongation: RNA Polymerase is Prossessive
RNA polymerase cannot let go of DNA unti lit has finished making the complete RNA transcript
44
Transcript Temination: Rho Dependent Termination
- CA rich region
- Rho is a Helicase (cousin of DNA B)
- Rho travels 5'-3' direction until catches up to polymerase
- Rho Travels along RNA using ATP hydrolysis
- It cannot catch RNA Polymerase unless it stalls (paused)
- RNA becomes hairpin and then RHO catches up
45
Transcript Temination: Rho Independent Termination
- Forms hairpin structure
- when hairpin forms, RNA polymerase stops and disocciates
- DNA sequence is followed by a string of 3 A residues, which are transcribedinto Uridylates at 3' end of RNA strand
- At this point, polymerase PAUSES bc hairpin affects association bw RNA pol and transcript
46
Eukaryotic RNA Polymerases
• Eukaryotes have three types of RNA polymerases which form
distinct complexes, although they do share some subunits.
• RNA polymerase I only synthesizes the precursors of ribosomal
RNAs. (FOCUSES ON rRNA)
• RNA polymerase II synthesizes mRNA precursors. It can recognize
thousands of promoters of varying sequence, through associated
proteins.
• RNA polymerase III synthesizes precursors of ribosomal RNA,
tRNAs, and other small RNAs.
-Proteins are required for Initiation of Transcription at the RNA Pol II called TF
47
Eukaryotic RNA Polymerases: RNA Polymerase II
- at least 12 subunits
- requires an additional array of transciption factors to form active transciption complex
- RBP1 subunit similar to B'- has a long tail with a sequence important for regulation
48
Eukaryotic RNA Polymerases: RBP1 Tail
Has alot of proline- so it'll probably a a loop.turn.
Also has TYR, SER, THREONIN- All hydroxyl groups, great for phosphoylation
49
Eukaryotic RNA Polymerases: Elongation Factors
Required for transcription
50
Initiation Protein: Pol II
catalyzes RNA synthesis
51
Initiation Protein: TBP (TATA- binding protein)
Specifically recognizes TATA box
52
Initiation Protein: TFIIA
Stabilizes binding of TFIIB and TBP to the promoter
53
Initiation Protein: TFIIB
Binds to TBP-recruits Pol II-TFIIF complex
54
Initiation Protein:: TFIIE
Recruits TFIIH; has ATPase and helicase activities
55
Initiation Protein: TFIIF
Binds tightly to pol ii, binds to TFIIB and prevents binding of POL ii to nonspecific DNA sequences
56
Initiation Protein: TFIIH
Unwinds DNA at promoter (helicase activity); phosphorylates pol ii; recruits nucleotide-excision repair proteins
57
Eukaryotic Promoter for Transciption:
- TATA at -30 instead of -10
| - Space can be several 1,000 bp away from the TATA box- which wraps around histones to attach complex
58
Eukaryotic Transciption: Steps
- assembly
- initiation
- elongation
- termination
59
Eukaryotic Transciption: Assembly
-begins when the TATA-binding protein
binds to the TATA box.
-TFIIB, TFIIF-RNA
Polymerase II, TFIIE, and TFIIH. are recruited to this site
60
Eukaryotic Transciption: Assembly- TFIIF
TFIIF is essential for guiding RNA Pol II to the
| correct DNA sequences.
61
Eukaryotic Transciption: Assembly- TFIIH
TFIIH binding completes the closed complex and
TFIIH starts to unwind the DNA to create the
open complex.
• TFIIH is also responsible for phosphorylating
RNA Pol II numerous times in its C-terminal
domain, causing a structural change and
initiating transcription.
--TFIIH is a multifunctinoal protein when it binds it completes the closed complex
AND unwinds the DNA by acting as a helicase AND serves as a kinase- phosphorylates RNA polymerase II numerous time in the C-terminal domain
-With phosphorylation, it drives conformaitonal change in the proteins (because of addition of negative charges) initiating trancription.
62
Eukaryotic Transciption: Assembly- TFIIE and TFIIH
TFIIE and TFIIH are released as RNA Pol II
synthesizes the first 60-70 nucleotides and
enters the elongation phase.
63
Eukaryotic Transciption: Assembly- TFIIF
TFIIF remains associated with RNA Pol II
throughout elongation. Additional elongation
factors bind to the complex to enhance the
activity
64
Eukaryotic Transciption: Termination
Termination involves dephosphorylation of RNA
Pol II, but the entire mechanism is not well
understood.
65
RNA Polymerase Inhibition: Acinomycin D
```
Actinomycin D intercalates into
successive G-C base pairs of
eukaryotic and prokaryotic DNA,
deforming the DNA and preventing
movement of RNA polymerase.
```
66
RNA Polymerase Inhibition: Rifampicin
Rifampicin binds to the BETA subunit of
bacterial RNA polymerases and
prevents extension past the promoter
67
RNA Polymerase Inhibition: Amanita Phalloides
```
The mushroom Amanita phalloides
makes a compound known as ALPHA-
amanitin which blocks RNA Pol II, but
not bacterial RNA polymerase. This
prevents predators from destroying the
mushroom population.
```
68
Post Transciptional Processing (not really because processed in real time)
These modifications take place in an organized fashion and are also
coordinated with transfer of the RNA transcript from the nucleus to
the cytosol.
-Eukaryotic transcripts generally contain the information for a single
gene, but they also have intervening noncoding sequences. These
sequences have to be spliced out of the RNA transcript. (INTRONS)
-some prokaryoutes have introns but are transposons that have to be processed as well.
69
Post Transciptional Processing (not really because processed in real time): Ribosome Function
RNA polymerase are making transcript and while that’s happening, Ribosome hops on
-Prokaryotes are going to have translation at the same time they are doing transcription
SO. RNA pol is making transcript and as transcropt it there, ribosome jumped on it and began making protein
70
Post Transciptional Processing (not really because processed in real time): Polysistronic mRNA
exists in prokaryotes, not common in Eukaryotes bc you need a 5' cap to define start site for protein biosynthesis.
71
Post Transciptional Processing (not really because processed in real time): Cap consisting of a 7-methylguanosine residue
-A cap consisting of a 7-methylguanosine residue is added to the 5’
end of mRNA transcripts
72
Post Transciptional Processing (not really because processed in real time): Poly A Tail
-The 3’ end has to be cleaved and a poly(A) tail of 80-250
| nucleotides is added.- uses Anenylate residues
73
Post Transciptional Processing (not really because processed in real time): Location of Trancription in Eukaryotes and Prokaryotes
- In PROKARYOTES: transciption and traslation are both happening in Cytosol
- In Eukaryotes: mRNA has to leave nucleus and go into the cytosol for translation
74
Capping of the 5' End: What is the cap?
- 7 Methyl guanisine
| - Added on the 5' residue of the newly synthesized RNA
75
Capping of the 5' End: Methyl Group is added to...
the first and possibly the second nucleotide of
the mRNA.
-purpose of methyl group:to enhance association with ribosome, lower Kd
76
Capping of the 5' End: when is it added?
The cap is added early in transcription and
comes from a molecule of GTP which forms a
bond with the 5’-end of the mRNA residue.
77
Capping of the 5' End: Where is it methylated?
• The cap is methylated at N-7 after it has been added to the mRNA
78
Capping of the 5' End: What enzyme synthesizes the CAP?
The enzymes that synthesize the cap are
| tethered to the polymerase CTD.
79
Capping of the 5' End: What does Capping mark?
Capping likely marks the completion of RNAP
II’s switch from transcription initiation to
elongation.
80
Capping of the 5' End: How is the cap bound to the polymerase complex?
• The cap is bound to the polymerase complex by
| the cap-binding complex.
81
Capping of the 5' End: Purpose of CAP
-The cap appears to help protect the mRNA from
ribonucleases and also participates in binding to
the ribosome (involved in translation)
82
When is the Cap methylated?
Cap is methylated AFTER added to RNA
| -Tail indicated RNA polymerase
83
Capping of the 5' End: Phosphodyhydrolase
5' end of RNA-- N is residue and P is phosodiester bond
- Attacking Gamma phosphorous atom with water (kicking off the rest of RNA)
- Gamma gets liberated as inorganic PHOSPHATE because it has the OH from water
- So you are working with DI phosphate not TRI
84
Capping of the 5' End: Granyltransferase
- activates O- from Beta phosphorous (nucleophile) and hit the alpha phosphorous atom in GTP (electorphile) that kicks off Beta Gamma (inorganic pyrol phosphate comes off from GTP)
- 5 prime 5 prime triphosphate bond is bonded
85
Capping of the 5' End: Guanine-7-methyltransferase
- Places methyl group on 7th position of guanine
- Gets it from AdoMet (AKA SAM) source of methyl group **Look up SAM and where methyl group comes from)**
- SAM becomes ADOHCY (SAW)
- 7 methyl guanisine is formed
86
Capping of the 5' End: 2' O-Methyltransferace
Remember: sometimes 2' is methylated
-Methyl is from SAM
-transferred over to 2' hydroxyl
(this can happen a couple times)
87
Capping of the 5' End: CPC (cap binding complex)
tethers 5' end to the RNA polymerase
88
Posttransciptional Processing: Poly (A) tails
Mature eukaryotic mRNAs have poly(A)
tails of 80-250 nucleotides appended to
their 3’ ends.
-Poly A tail is needed to have RNA associate with Ribosome
-Need poly A tail to make transcript translated to make a protein, length of tail correclates withh lifetime of RNA
-half life reflected by tail length
-long tail-> transcipt stays around of a long time
- AAUAA (recognition sequence/ Signal Sequence)- what triggers machinery to start laying Poly a tail down.
- Tail protects Eukaryotic mRNAs and cause degredation of bacterial mRNA
-Prokaryotes DON’T like A tail
Poly A polymerase: proteins dictate how long
89
Posttransciptional Processing: Endonuclease
The mRNA molecule is first trimmed
back to within ~20 nucleotides of the
AAUAAA sequence by an endonuclease.
90
Posttransciptional Processing: Within Enzyme Complex
Within enzyme complex: Endonuclease, poly A Polymerase, proteins involved in sequence recognition, proteins that stimulate cleavage, regulation of tail length.
- enzyme comlex is going to recognize a signal sequence and bind to it. What happens when it binds?-
- Endonuclease activity is fired up (cuts transcript to stretch of 20) and RNA polymerase leaves
91
Posttransciptional Processing: Exonuclease
```
The lifetime of most mRNAs is on the
order of hours to days. The poly(A) tail
shortens over time and transcripts that
lack poly(A) tails are degraded in <30
minutes. Exonuclease shortens it.
```
92
Intron VS Exon
• The coding part of a gene is known as an exon, while
non-coding parts are known as introns.
• Eukaryotes tend to have many introns.
• Exons tend to be shorter in length (~150 nts) than introns
(~3500 nt). Introns must be removed from the final
mRNA product before it is transported to the cytosol.
93
4 Major groups of Introns
- Group 1 and Group 2
- Spliceosomal Intron
- tRNA Intron
94
Group I and Group 2
introns are self splicing
– Interrupt mRNA, tRNA and rRNA genes
– Found within nuclear, mitochondrial, and chloroplast genomes
– Common in fungi, algae, and plants, also found in bacteria
– Group I and Group II differ mainly by the splicing mechanism
95
Spliceosomal Intron
are spliced by spliceosomes
– These are most common introns
– Frequent in protein-coding regions of eukaryotic genomes
96
tRNA Intron
are spliced by protein-based enzymes
– Found in certain tRNAs in eukaryotes and archae
– Primary transcript cleaved by endonuclease
– Exons are joined by ATP-dependent ligase
97
Group 1 Introns
```
use
GMP, GDP, or GTP (GUANILATE) to
initiate the splicing
process.
• The nucleotide attacks
the 3’-end of the first
exon, generating a 3’-
OH that will then attack
at the 5’-end of the
second exon.
• The intron is then
released and
eventually degraded
```
Group 1 Introns DO NOT appear to have consensus splice site
LOOK AT PIC
98
Group 2 Intron
```
Group 2 and Splicsosomal enzymes DO have consensus splice sites
-Uses 2'OH as nucleophile
-Group II introns use an
adenosine residue within
the intron to initiate
splicing.
• A branched lariat
structure is formed as an
intermediate, and then
released.
```
LOOK AT PIC
