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Mol. Cell Bio > Ch 11: Expression of Genetic information > Flashcards

Ch 11: Expression of Genetic information Flashcards

1
Q

Central dogma of molecular biology

A

DNA -> RNA -> protein

not necessarily true, RNA -> DNA is possible through reverse transcriptase

and RNA can have catalytic function i.e. Ribozymes

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2
Q

Alkaptonuria

A

A disease studies by Garrod in 1908 that is characterized by dark urine.

Trait is ingerited (genetic basis)

Affected indiciduals are deficient for enzyme responsivle for oxidizing homogentisic acid (part of the pheylalanine breakdown pathway)
enz = homogentisic acid oxidaseq

invented term “inborn error of metabolism”

one gene-one enzyme hypothesis is derived

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3
Q

Beadle and Tatum experiment

A

irradiated neurspora spores to induce mutations

gre irradiated neurospora on complete medium

Gre individual spores on minimal medium to identify mutants

mutant smaples tested on minimal medium supplemented with specific vitamins

by growing on minimal medium, wwyou can see which enzyme // metabolite is missing, or cannot be converted

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4
Q

Vernon Ingram and sickle cell anemia (1956)

A

used proteolytic enzymes = trypsin to cleave normal and sickle cell hemoglobin polypeptides (hemoglobin A vs S)

identified fragments with two dimensional paper chromatography (seperation via charge [left-right] and molecular size [up-down])

one fragment migrated differently

sequenced fragment and identified mutation

one gene-one protien/// polypeptide

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5
Q

Sickle cell Beta Globin Protein

A

Glu (-ve charge) to Val (neutral) aa sub

results in sticky patches on moleucle

form crystals that distort rbc

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6
Q

flow of genetic informaiton

A

use of mRNA separates storage of genetic information form information utalization

also permits amplification of gene information

proteins translated from mRNA on ribosomes

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7
Q

types of RNA

A

mRNA

rRNA

tRNA

tRNA and rRNA form secondary structures

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8
Q

mRNA

A

transcribed form DNA template

code for protien

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9
Q

rRNA

A

structural and catalytic RNA in ribosomes

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10
Q

tRNA

A

carries aa ro ribosome and mRNA transcript

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11
Q

Groupo II self-splicing intron

A

RNA secondary strucutre

has selfsplicing activity

hairpin loop exhibits ribozyme acticity

proximiy effects increase rate

hydroxy group acts as a nucleophile (A) to attack (G)
then 3’ end hydroxy group acts as a nucleophile to push off the intron= leaving group

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12
Q

Basics of transcription

A

RNA transcribed by DNA dependent RNA polymerase (DNA template to make their transcripts)

Polymerase binds to promoter (upstream of the gene coding region) with aid of transcription factors

DNA temporarily unwound as polymerase moves along (by the aqid of helicases and topomerases)

Mg2+ is required in the active site as a cofactor

RNA is trancribed in the 5’ to 3’ direction

transcription bubble is 35 bp of DNA, DNA-RNA hybrid is 8 bp

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13
Q

Transcription elongation

A

DNA clamped down by RNA pol

(and twisted???? see slide 24) ch 11

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14
Q

Direction of RNA transcription

A

RNA synthesized in the 5’ to 3’ direction

pyrophosphate is hydrolyzed to make the overall process favourable

3’ OH of nucleotide at the end of the growing strand attack 5’ alpha phosphate of the incomping nucleotide triphosphate

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15
Q

Experimental system for studying RNA transcripton

A

RNA ligated to glass surface by a his tag

fluorescent bead is on the upstream end of DNA, can measure wobble

fluoresent bead is on the downstram end of DNA, can measure the force produced by the enzyme

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16
Q

RNA has high processivity

A

stays associated with DNA remplate with high efficiency

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17
Q

RNA produces force while transcribing

A

force produced by RNA pol is twice that of myosin moleucle

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18
Q

How is energy supplied to RNA pol

A

energy derived from the hydrolysis of NPPP = ribonucleotide precursors

pyrophosphate is hydrolyzed

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19
Q

RNA pol movement

A

movement is not cts

polymerase may stall
=fix mistakes

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20
Q

RNA pol has high fidelity

A

RNApol backtracks to correct errors, and cleaves the mishap

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21
Q

Look at the video of RNA pol backtracking

A

on nexus website

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22
Q

sense strand

A

coding strand

in ds DNA that carries the translatable code i the 5’ to 3’ direction

this strand has the same sequence of mRNA

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23
Q

anti-sense strand

A

responsible for the RNA that is later translated to protein (is the template during transcription)

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24
Q

Initiation of transcription in prokaryotes

A

core enz + sigma factor to help increase the core enzymes affinity for the promoter active site

this does not stay associated during transcription once RNA pol starts, sigma factor leaves.

-35, -10 upstream of the initiation site

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25
Bacterial promoters
located in the region preceding transcription initiation 35 bases upstream TTGACA consensus sequence =recognized by sigma factor 10 bases upstream TATAAT\ =pribnow box, is important for proper alignment, such that it can start transcription aty the start site
26
Comparison of RNApol
archeal and eukaryotic RNA pol are more similar to each other than either is to bacterial polymerase
27
Eukaryotic RNA pol
RNA pol I RNA pol II RNA pol III RNA pol IV
28
RNApol I
synthesizes large rRNA transcripts 28s, 18s, 5.8s
29
RNApol II
synthesizes mRNA and small nuclear RNA (snoRNA and snRNA)
30
small nucleolar RNA
snoRNA
31
small nuclear RNA
snRNA
32
RNApol III
synthesizes tRNA and small rRNA 5s rRNA
33
RNApol IV
synthesizes siRNAs plants only
34
Nucleolus, the site of Ribosome assembly
darker bodies with in the nucleus gc = granular component = ribosomal subunits in process of assembly fc = fibrillar component = DNA (genes) coding for rRNA (ribosomal RNA) dfc = dense fibrillar component = rRNA nascent transcripts
35
Synthesizing rRNA precursor
rRNA genes in oocytes are amplified during development increase number of nucleoli expand tandem arrays of rRNA genes via rolling circle replication
36
Rolling circle replication
making RNA transcripts circular DNA makes many linear copies can re-insert themselves back into the genome
37
rRNA transcription unit
5s in a differnet part of the genome but these three = 18s, 5.8s and 28s are transcribed together needs to be processed post transcriptional processing (much different than RNA pol II which is one gene, one transcript)
38
kinetic analysis of rRNA synthesis and processing
pulse chase exp I have no fucking clue see lec jan 25
39
what is the first mature transcript ot be seen in the cytoplasm with mammalian rRNA
18s this is later followed by by the 28s and the 5.8s
40
modification of mammaliam rRNA
large number of methylated nucleotides and pseudouridine residues in processed rRNA (post translation modification are conserved, that is, they are always on the same residue) =posttranscriptional modificaiton = conserved regions ex =- methylation
41
Roles of modified residues in the modification of mammalian rRNA
protect from cleavage promote folding promote interactions with other molecules (proteins)
42
role of snoRNAs
small nucleolar RNAs complex with protein to form snoRNPs (a RNA/ protein combintion) Antisense snoRNAs = U3 BOX C/D BOX H/ACA
43
Antisense snoRNAs
bind to RNA help modification enzymes recognize rRNA (methylase)
44
BOX C/D snoRNAs
= methylation guide RNA thats responsible for methylating uracil residures bind to RNA and helps modificaiton enzymes (methylase) recognize rRNA antisense snoRNA
45
5s rRNA processing
genes are seperate from other rRNA genes (unlike the other rRNA transcripts) located outside the nucleolus organized in tandem arrays transcribed by RNA pol III => promoter is internal (w/ in the gene, not upstream) 5s rRNA transported back io nucleolus following processing 5s rRNA is first to be seen because it doesn't have the extra processing the other transcripts have
46
tRNA processing
~50 different species of tRNA (b/c need redundancy) => many codons for the same aa (helps with silenct mutations??) found in clusters throughout genome transcribed by RNA pol III => internal promoter Pimary transcript is processed (RNase P)
47
Bacterial tRNA processing
Ribonuclease P is needed for proper post translation modifivations of tRNA good example of the RNA being catalytic// ribozyme cleaves off the 5' end
48
mRNA transcription
mRNA transcripts transcribed by RNA pol II initiation depends on transcripiton factors promoters are upstream of coding sequence => core promoter element between 24 and 32 bases upstream => TATA box is the site of formatio of initiation complex
49
Know the formation of the preinitiation complex
know it many general transcription factors (GTFs) cause all genes use these for the pre-initiation complex (which is before RNA pol II transcribes) TAF DPE TBP TFIIB TFIIF TFIIH help align and dock RNApolII so it aligns at the proper start site
50
TAF
TATA binding protein associated factors
51
DPE
downstream promoter element
52
TBP
TATA binding protein bends DNA approx 80 degrees and allows TFIIB to bind does this via insertion into the minor groove of DNA helix
53
TFIIB
provides bindng site for RNA pol II
54
TFIIF
contains subunit homolgous to bacterial sigma factor
55
TFIIH
is a multisubunit protien with enzymatic proterties (kinases) phosphorylation of RNA pol II promotes the uncoupling of enzyme from the promoter region of RNA pol II rich in serine is phosphorylated = CTD = C terminal domain
56
CTD of RNA pol II
C terminal domain is rich in serine and is phosphorylated by TFIIH which can allow it to be uncoupled form TFs and begin transcription
57
mRNA structure
encode a specific polypeptide found in cytoplasm attached to ribosomes contain noncoding segments (Untranlated UTR = untranslated region) eukaryotic mRNAs are modified = 5' methyl guanosine cap = 3' poly A tail
58
Discovery of split genes
proposed that hnRNA =heterogenous RNA was processeed to form mRNA discovered that mRTTNA is transcribed form segmetns of DNA Discovered intervening sequences in adenovirus genome = introns R-loop formation experiments used to locate intervening sequences
59
U3 snoRNA
binds to the 5' terminus and helps to catalyze the removal of 5' end of transcript
60
Box H/ACA snoRNA
conversion of uridine to pseudoeuridine align themselve and form a hairpin opposite of the nucleotide to be modified
61
hnRNA
nacent RNA from DNA transcription =primary RNA transcript done associated with ribosomes, ribosomes only associated with mature RNA =mRNA hnRNA is much longer than mRNA and is extremely variable in size. -> this is opposed to the average size of mRNA trancscripts ~2kb
62
Restriction map changes
allows us to tell if RNA is modified smaller transcripts = modifications have to look at cDNA because you can use restriciton enzymes on RNA
63
visualizing introns r-loop formaiton experiments
Displaced single stranded DNA implies mRNA exon. Sense strand binds and displaced antisense-template strand of DNA. Displaced dsDNA implies introns have been removed. Thus, there is no complimentary strand for the mRNA to displace so the DNA bulges out and forms a loop.
64
TIming of mRNA transcript processing
co-transcriptionally Pre-mRNA transcripts are processed as they are synthesized
65
mRNA modificaton
Capping and polyadenylation For capping, the removal of the last of the three phosphates on the 5' end by RNA triphosphatase is performed first. Then, a guanine is added on its 5' end by guanylyltransferse by a tri-phosphate bridge. The 2' positon of guanine is then methylated by RNA methyltranserase =Methylguanosine cap For polyadenylation, the 3' end is first cleaved, and then Poly(A) polymerase can add adenosines redidues via non-template addition for about 250 residues.
66
intron types
Group 1 Group 2 Spliceosomal intron
67
Group 1 introns
self-splicing introns first discovered in tetrahymena these form complex sencondary structures
68
Group 2 introns
self splicing introns found in fungal mitochondria and plant chloroplasts use a lariate intermediate for splicing also have that branch point adenine -> performs a nucleophilic attack via its 3' hydroxyl towards the 5' end of the intron. This makes the lariet, which then will perform a secondary nucleophilic attack on the 3' splice site. Ribonuclease activity
69
Spliceosomal introns
found in animal cells use a lariate intermediate + nuclear snRNAs U1 snRNP attaches at the 5' branch site (btwn exon and intron). U2 is recruited by U2AF, and attaches at the adenine branch site. U1 is displaced by U6 (which has ribozyme activity), and U4 dissociated from the U4/5/6 complex. Together U6 and U5 have catalytic helicase activity. cleavage of the 5' splice site by U2/ U6 while U5 holds the exons in proximity. Second cleavae reaction at the 3' splice site frees intron, and exons are ligated together.
70
Ribonuclease P
processign of tRNA precursors in bacteria pTyr -> Tyr and 5' Tyr 23 S rRNA of prokaryotic ribosomes
71
Spliceosomal vs. self-splicing introns
conformationally and mechaninally similar. Spliceosome evolved from a self splicing intron?
72
CTD of RNApol 2
CTD serves as a scaffold (when is serines are phos) for splicing, caping and polyadenylation. recall, modificaitons of RNA transcripts begin before transcription ends
73
Origin of introns
see introns early vs introns late article on nexus introns introduced by endosymbionts introns became self splicing exons (catalytic portions) became snRNA genes introns become variable in size
74
Advantages of introns
alternative splicing adds diversity to genes snoRNAs encoded in ribosomal protien introns Exon shuffling - > introms are potential recombinations sites = genetic varation - > recombination increases b/c more DNA exists - > Better shuffling
75
Alternative splicing
depends on snRNA avalible in the tissue exon skipping/ inclusion alternative 3' spice sites alternative 5' splice sites mutally exclusive exons intron retention (intron is left in, can occur in the intron is in the reading frame)
76
PLC and exon shuffling
PLC is a parts bin exons code for individual protien domains, and PLC is compled of components of four distinct proteins
77
RNA interference
double stranded RNA sequences capable of destructon of select mRNAs -> recoignize mRNA with complementary sequence may be a primitive immune system Double stranded RNA cleaved into small interfering RNAs (siRNA) by DICER enz. antisense strand incorperated into protein complex RISC leads to destruction of complementary mRNA by RNAi See slide 107????
78
Process of siRNA
dsRNA synthesized DIcer enzyme cleaves dsRNA (chops up with little overhangs ->dsRNA fragments) This is incorperated into RISC complex associates RISC complex associates and unwinds RNA in ssRNA (antisense to target mRNA) siRNA binds to mRNA target mRNA cleaved (endonuclases avtivty)
79
micro RNAs (miRNA)
small RNA sequence complementary to 3' UTR of specific mRNA (first discovered in C. elegans) -> sequence is highly conserved, miRNAs conserved btwn divergent organsims synthesized at specific times during development - > may turn genes on and off - > translational inhibitors (instead of degredation, they inhibit ribosomal association)
80
process of miRNA
single stranded RNA folds back on itself cleaved by dicer enzyme RISC complex associates miRNA binds to mRNA target block translation
81
role of miRNAs
developmental programming patterning of nervous system control of cell proliferation and cell death cell differentiation
82
Lin-4
in 1993 it was discovered that C. elegans lacking lin-4 gene was unable to develop normally lin-4 encodes small RNA complementary to 3' UTR of mRNA encoding the protien LIN-14 lin-4 mutants possess high level of LIN-14 protien
83
Let-7
highly conserved found in both worms and humans let-7 mutant animals die by bursting through the vulva at the L4 to adult moult
84
siRNAs vs miRNAs
siRNAs derived from double stranded product of virus of synthetic dsRNA miRNAs encoded by genomic region miRNAs target specific mRNA transcripts miRNAs primary role is to regulate gene expression
85
si-RNA from run on gene transcription
si can be produced in vivo when run-on transcription occurs extra mRNA transcripts can hybridize to form dsRNA
86
piwi RNA
expressed in germ cells suppress movement of transposable elements associate with PIWI protiens required for successful gamete formaiton -> transposible elements SHOULD NOT be allowed to be active in germline cells Deletion of PIWI protiens leads to failure in gamate formaiton
87
CRISPER
found in bacteria, clustered repeats are remnants of bacteriophage genomes CRISPER RNA transcript cut into smaller guide sequences associate with cas9 protein -> targett and cleave DNA with homology to guide RNA (primitive immune system) can use to edit genes
88
transcriptome
at least 2/3 of the mouse and human genome is transcribed this is way more than would be expected, since lots of it is non-coding transcripts overalap and originate from both sense and antisense strands debate between wheiter they are junk or functional transcripts
89
junk transcripts
loss of regions coding for transcripts does not seem to affect development
90
functional transcripts
have reproducible patterns of tissue specific distribution
91
Deciperhign the code of DNA/RNA
nirenberg and philip leder developed a technique using ribosome-bound transfer RNAs (tRNAs) synthesized short 3 nucleotide mRNAs incubated with ribosomes and labeled charged tRNAs identified with tRNA/ aa bound to syntheic mRNA
92
third position degeneracy
tRNA wobble
93
Purpose of clustering codons for AA
single base mutation will not always cause the change of codon (synonymous mutation) nonsynonymoys mutations change AA coding excess of nonsynonymoys to synonymous mutations suggests a selective advantage similar AA clustered together in chart
94
three phases of translation
initiation elongation terminaiton
95
Translation initiation
ribosome attaches at initiation codon (AUG), this helps to establish a readign frame Step 1 = small ribosomal subunit associated with initiation codon, aligned by -25 shine dalgarno sequence GGAGGA on mRNA
96
step 1 of prokaryotic translation initiation
this is the process of bringing small subunit to initiation codon IF2: required fo rthe attachment of the first aminoacyl-tRNA IF3: prevents large subunit (50s) from joining. Facilitates entry of the intial aa-tRNA IF1: facilitates attachemnt of 30s subunit to mRNA and prevents aa-tRNA from entering wrong area
97
step 2 of prokaryotic translation initiation
bringing first aa-tRNA into the ribosome enters P site and binds to AUG codon and IF2 initiation factor IF3 and IF1 releases
98
step 3 of prokaryotic translation initiation
assemblind the comple initiation complex large subunit joins the complex and GTP bound to IF2 is hydrolyzed
99
Translation initiation in in eukaryotes
eIF2-GTP => associates with initiator tRNA eIF4G => links 5' cap and the poly A tail eIF4E => binds to the 5' cap eIF4A => removes double stranded regions
100
step 1 of prokaryotic translation elongation
aminoacyl-tRNA selection aminoacyl tRNA enters the A site
101
step 2 of prokaryotic translation elongation
peptide bond formaiton peptidyl transferase activity in large subunit amino group of aa-tRNA reacts with carbonyl group of P site
102
step 3 of prokaryotic translation elongation
translocation deacylated tRNA moves to E site binding of EF-G and hydrolysis of GTP results in translocation of ribosome
103
step 4 of prokaryotic translation elongation
releasing the deacylated tRNA
104
Ribosome oscilatates between two states during the process of elongation
hybrid stats of transslocation observed by cryo-microscopy tRNA anticodons still reside in A and P sites of small subunit while acceptor ends move to P and E sites (A/P and P/E) EF-G stabilizes ratcheted state and prevents movement back of tRNAs back to A/A and P/P conformation GTPhydrolysis causes conformational change that moves mRNA and tRNAs to E/E and P/P sites
105
Translation termination
termination codons => UUA, UAG, UGA Requires release factors in prokaryotes, the release factors are: - >RF1 recognizes UAA and UAG - >RF2 recognizes UAA and UGA - > RF3 not codon specific in eukaryotes: -> eRF1 and eRF3 combine and recognize all termination codons
106
mRNA survaillance
looks for mRNA with premature stop codons nonsense mutations detected by specific process => aka nonsense mediated decay pathway exon-junction complex normally displaced by advancing ribosomes => these complexes remain on transcripts with premature stop codons since they will not be knocked off my RNA polymerase (ribosome reaches stop codon and falls off before it reachs the EJC protiens complex)
107
Polyribosomes
more than one ribosome associated with a transcript this helps to increase the rate of protien synthesis

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