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week 7 Flashcards

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1
Q

nucleosomes

A

DNA packaged in eukaryotes by organising DNA into chromatin using nucleosomes

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2
Q

histone proteins

A

H1- small, basic
H2A- (+ve) proteins
H2B- H3, H4- very highly conserved

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3
Q

chromatin

A

DNA + histone proteins
DNA tightly packaged to fit in nucleus of cell

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4
Q

core nucleosome

A

contains 2 copies of H2A, H2B, H3 and H4- DNA wound round outside of core nucleosome
H1 associated with DNA between nucleosomes- linker histone forming ‘beads on a string’ structure

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5
Q

introns

A

interrupt coding region of eukaryotic genes
absent in final mRNA
transcribed from DNA into RNA but removed by a processing reaction to generate final mRNA

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6
Q

exons

A

present in final mRNA

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7
Q

RNA polymerase II

A

transcribed DNA, if the gene is a protein coding gene
has 10+ subunits
needs general transcription factors and activator proteins to find promoters and to at transcription at a high lvel

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8
Q

polyadenylation site

A

marks end of mRNA product

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9
Q

basic structure of eukaryotic gene

A

RNA starts at promoter region
protein start codon (ATG)
- not the same as RNA start point)
introns and exons along gene
protein stop codon present (TAG, TAA, TGA)
- not same as RNA end point
polyadenylation site at end of RNA
RNA polymerase II moves from 5’ untranslated region to 3’ untranslated region

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10
Q

primary transcript

A

contains introns as well as extra sequence at 3’ end (beyond polyadenylation site)

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11
Q

processing reactions

A

remove extra region at 3’ end back to polyadenylation site
introns and exons joined together to produce final mRNA
known as RNA splicing
only exons present in final mRNA

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12
Q

transcription

A

occurs in nucleus

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13
Q

translation

A

occurs in cytoplasm

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14
Q

roles of proteins in transcription

A

RNA polymerase I - rRNA
RNA polymerase II- mRNA
RNA polymerase III- tRNA, snRNA, 5SRNA
general transcription factors- promoter location
activator proteins- stimulation of transcription

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15
Q

core promoter

A

region where transcription starts
contains RNA initiation site
TATA box present in some which is 25bp upstream of RNA start site

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16
Q

proximal promoter

A

next 100-200bp or so upstream of core promoter

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17
Q

enhancer

A

often many 1000s of bases upstream
plays a role in regulating transcription from the core promoter
DNA elements recognised by proteins (TF and activators) that target RNA polymerase II (this cannot bind without help

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18
Q

general transcription factors

A

control the transcription process
interact with DNA at core promoter
present in all cells and forms the transcription ‘machine’ in combination with RNA polymerase
assemble a transcription complex at the core promoter

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19
Q

activators

A

interact with DNA at the proximal promotor and enhancer
may be tissue specific
regulate the level and timing of transcription of individual genes
long way from CP- up to 1 mil bases in mammals
- could interact and influence CP by the DNA between the enhancer and CP looping round so the proteins attached to the enhancer make contact with the general TFs assembling at the CP

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20
Q

stepwise assembly model

A

GTF are TFIIA, TFFIIB, TFIID, TFIIE, TFIIF
assemble in order at the core promoter to allow RNA polymerase II to associate

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21
Q

process of GTF assembling

A

TFIID is first TF to bind
- mix of proteins- TBP and several TAF proteins
TBP binds in minor groove of DNA and brings TFIID into the core promoter
then TFIIA, then TFIIB, then TFIIF and RNA polymerase II, then TFIIE, then TFIIH
assemble in defined order on CP to establish transcription complex

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22
Q

activators with TC

A

influence the assembly of the TC and structure of chromatin
stimulate transcription in 2 ways:
- make assembly of the T more efficient
- unpack the chromatin to allow transcription

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23
Q

roles of mRNA cap

A

protects 5’ end of the mRNA from degradation and is important for mRNA stability
cap interacts with TF which recruit ribosomes for protein synthesis
cap interacts with the cap binding complex involved in export of mRNA from the nucleus
cap may be importance in the splicing of introns near the 5’ end of the mRNA

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24
Q

polyadenylation at the 3’ end

A

sequence AAUAAA in the mRNA targets a protein complex
endonuclease complex cuts the mRNA 11-30 bases past the AAUAAA
removed part of the RNA is degraded
remaining mRNA is polyadenylated
poly A tail is important in determining mRNA stability in and in aiding translation

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25
3' end of eukaryotic mRNA
5' end of mRNA corresponds to the position where transcription starts at 3' end, transcription continues past the position of the end of the mRNA to make a longer RNA which is then cut and polyadenylated 3' end of mRNA is generated by cleavage
26
endergonic nature of polymerisation
driven by "high-energy" phosphoanhydride bond cleavage
27
genetic code
'codon' is necessary to specify a single amino acid triplet code is sufficient to specify upt o 20 AA types triplet code allows amino acids to be specified by more than 1 codon (it is degenerate) code is non overlapping
28
experimental basis for genetic cod
Crick and Brenner used bacteriophage T4 - discovered that deletion of a nucleotide could abolish gene function and a 2nd mutation could restore gene function - mutations are 'suppressors' of one another concl: genetic code is read sequentially from a fixed point- insertion or deletion of an AA shifts the 'reading frame' insertion or deletions known as frameshift mutation further investigation showed that two closely spaced mutations could not restore gene function, but 3 could therefore proving triplet nature of code
29
deciphering genetic code
mRNA does not directly recognise AA - binds molecules o tRNA (carrying AA) - each tRNA contains an 'anticodon' complementary to mRNA codon AAs on tRNAs are joined together in order that tRNA binds to mRNA NDPs are linked at random so that base composition of product reflects reactant NDP mix
30
use of cell free system
E.coli cells broken open and centrifuged to remove cell walls (left with DNA, mRNA, ribosomes, enzymes) protein synthesised with ATP, GTP and AA added add DNase to remove the DNA add purified or synthetic mRNA - recover resulting polypeptide poly(A)= poly(lys) poly(C)= poly(pro)
31
promoting tRNA binding to ribosomes
trinucleotides were tested for their ability to promote tRNA binding to ribosomes ribosomes bound to nitrocellulose filter - free tRNAs pass through - bound tRNA is identified by the labelled AA attached UUU stimulated only Phe tRNA UUG= Leu tRNA binding UGU= cys tRNA binding GUU= val tRNA binding used to work out amino acids specified by 50 codons - for remaining codons, was either no binding or ambiguous
32
H Gobind Khorana experiment
chemically synthesised polynucleotides with repeating sequences In cell free translation system UCU CUC UCU stimulates production of Ser-Leu-Ser-Leu poly (UAC) specified 3 different homopolypeptides ribosomes may initiate polypeptides in any of 3 reading frames
33
start codons
AUG and GUG specify the starting point for polypeptide chain synthesis can also specify met and val at internal positions in the polypeptide chain
34
stop codons
UAG, UAA, UGA nonsense codons do not specify amino acids but signal translation termination
35
universal
studies in 1981 revealed genetic cods of certain mitochondria were variants of the "standard" genetic code alternate genetic code in ciliated protozoa as well - branched off early in eukaryotic evolution
36
Transfer RNA
tRNAs have similar structure - 54 to 100 nucleotides arranged in cloverleaf structure - 5'-terminal phosphate group - 7-bp stem ('acceptor') containing a 5'- terminal nucleotide and non-watson and crick base pairs - 'D-arm' which ends in a 7-nt loop containing dihydrouridine - 'anticodon arm'- 5-bp stem ending in a loop containing the anticodon - 'tψc arm' - 3'CCA sequence with free 3-OH
37
G-quadruplex
unusual secondary structure of DNA found in telomeres
38
variant arm
site of variability on tRNA consisting of 3 to 21 nucleotides tRNAs . 25% of post-transcriptionally modified bases - not essential for maintaining tRNA's integrity - promote attachment of AA to acceptor stem - strengthen codon- anticodon interactions
39
tRNA has complex 3⁰ structure
tRNA molecules have an L-shape - base pairing occurs between stems - acceptor and T stems form 1 leg D & anticodon stems form the other narrow: 20- to 25-Å width - 3 tRNA molecules bind in close proximity on adjacent codons
40
Aminoacyl- tRNA synthetases
selects the correct amino acid for covalent attachment to the correct tRNA it is AA-specific and attaches AA - 3-terminal ribose residue of tRNA - forms AA- tRNA
41
aminoacylation
catalysed by 1 enzyme in 2 steps 1) AA is activated - forms aminoacyl-adenylate 2) aminoacyl- AMP reacts with tRNA to form aa-tRNA - AA + tRNA + ATP --> aminoacyl-tRNA + AMP + PPi
42
affect of degeneracy on aminoacyl-tRNA synthetases
more than 1 tRNA may carry a spec AA - 'isoaccepting tRNAs' - must all be recognised by 'aaRS' synthetase contacts the tRNA in the acceptor stem and anticodon loop those that interact with the anticodon + acceptor stem must be able to bind both legs of the tRNA
43
yeast AspRS
homodimer of 557- residue subunits symmetrically binds 2 tRNA molecules conformation of a tRNA in complex with its cognate synthetase dictate by its protein interaction (induced fit), rather than by its sequenc e
44
proofreading DNA
tRNA charging is an accurate process tRNA synthetase subjects aminoacyl-adenylates to a proofreading step
45
'wobble'
protein synthesis requires the proper tRNA being selected for via codon-anticodon interaction each of the 61 codons are not read by different tRNAs- many tRNAs bind to 2/3 codons non-watson-crick base pairing can occur at the 3rd codon-anticodon position - normally contains Gm ( G with a 2'-methyl group) and I (Inosine)
46
how wobble accounts for codon degeneracy
first 2 codon-anticodon pairings= watson-Crick base pairing at least 31 tRNAs are required for translating all 61 triplets frequently used codons are complementary to the most abundant tRNA species
47
modus operandi
found at porton down an N-glycosidase from caster beans inactivated the large eukaryotic ribosome - by hydrolytically removing adenine base of 28S rRNA - so it is unable to bind to elongation factors - acts catalytically not sterically
48
ribosome
binds mRNA so that codons can be read includes binding sites for tRNA molecules mediates the interaction of non-ribosomal protein factors promoting: - polypeptide chain initiation - polypeptide chain elongation - polypeptide chain termination catalyses peptide bond formation allows translation of sequential codons to be capable of movement prokaryotic ribosomes (70S) consist of two subunits - small (30S): 16 S rRNA and 21 proteins - large (50S): 5S and 23s rRNA and 31 different proteins
49
ribosomal proteins
located on subunit back and sides not near tRNAs and mRNA binding sites stabilising function ribosome is a ribozyme
50
3 tRNA- binding sites
'A-site'- aminoacyl site - accommodates the incoming aminoacyl-tRNA 'P site' (peptidyl site) - accommodates the tRNA attached to growing peptide chain 'E site' (exit site) - accommodates the tRNA, without AA, that is leaving
51
tRNA binding
all 3 tRNAs have anticodons bound to 30S subunit, the rest of the tRNA bound to 50S subunit tRNAs on the A site and P site interact closely with mRNA via base-pairing - acceptor ends are close together (permits the peptidyl transferase reaction)
52
eukaryotic ribosomes
80S 40% bigger than bacterial version small subunit= 40S: 33 polypeptides + 18S rRNA large subunit (60S): 49 polypeptides, 28S, 5.8S, 5 rRNA
53
translation requirements
apart from the ribosome, the stages of translation (initiation, elongation and termination require factors for peptide synthesis - some are catalytic, others stabilise ATP and GTP used as energy sources - incl 2 from GTP - 1 for binding aminoacyl-tRNA to A site, 1 for translocation step
54
chain elongation
linkin g the growing polypeptide to the incoming tRNA's amino acid residue - growing polypeptide is transferred from peptidyl- tRNA in the P site to the incoming aa-tRNA in the A site new peptidyl-tRNA is transferred from A to P-site uncharged tRNA moves to E site
55
polysome complex
formed because of the lengths of mRNAs when more than one ribosome can translate a message
56
initiation
involves assembly of the translation system components before peptide bond formation includes: - 2 ribosomal subunits - mRNA to be translated - aminoacyl- tRNA specified by the first codon - GTP -initiation factors
57
ribosomes recognise the start codon
via 'shine-Dalgarno sequence' - binds with 16S rRNA of 30S subunit - not in eukaryotes - 40 S ribosomal subunit binds to cap structure 2. initiating AUG codon is recognised y special tRNA (gos straight to small subunit P site) - facilitated by IF-2-GTP in prokaryotes/ eIF-2-GTP in eukaryotes - larg subunit then joins complex
58
initiation
phase of translation involves the assembly of the translation system components before peptide bond formation
59
what does initiation phase include
2 ribosomal subunits mRNA to be translated aminoacyl-trNAspeciified by 1st codon GTP initiation factors initiating AUG codon is recognised by special tRNA- facilitated by IF-2-GTP in prokaryotes and eIF-2GTP in eukaryotes
60
shine dalgarno sequence
how the ribosomes recognise the start codon binds with 16S rRNA of 30S subunit not in eukaryotes - 40S ribosomal subunit binds to cap structure
61
elongation
phase of translation after initiation involves addition of amino acids to the carboxyl end of the growing chain delivery of the next aminoacyl-tRNA by EF: EF-Tu-GTP and EF-Ts requires GTP hydrolysis
62
elongation ii
ribosome advances 3 nucleotides to mRNA 3' end - also known as translocation translocation causes movement of uncharged tRNA from the P site to the E site peptidyl-tRNA from the A to the P-site
63
termination
termination codons recogniseed in e.coli by release factors : RF-1, RF-2 RF binding causes hydrolysis of bond linking peptide to tRNA in p-site protein is released RF-3-GTP causes the release of RF-1 and RF-2 as GTP is hydrolysed the ribosomal subunits, mRNA, tRNA and protein factors can be recycled
64
RF-1
recognises UAA, UAG release factor involved in translation termination
65
RF-2
recognises UGA, UAA release factor involved in translation termination
66
streptomycin
antibiotic which blocks translation member of aminoglycosides low- ribosome misreads mRNA, pyrimidines may be mistaken in the 1st and 2nd codon positions high- prevents chain initiation which causes cell death
67
chloramphenicol
1st gen broad spectrum antibiotic inhibits peptidyltransferase activity binds near a-site toxic side effects as it can affect mitochondrial translation
68
tetracycline
broad spectrum binds to small subunit of prokaryotic ribosomes prevents entry of aminoacyl-tRNA into A site allows EF-Tu to hydrolyse GTP - stops synthesis and is an energy drain
69
co and post-translational modification
ribosome-associated chaperones will help a protein to fold ribosome's exit channel is too narrow to permit 2 degree structure formation
70
cotranslational mod
polypeptide chain is modified before use and it is still attached to ribosome
71
posttranslational mod
polypeptide chain modified before use but after synthesis
72
pre proteins
secreted proteins are precursor proteins specialised endoproteases activate molecule - cleaved in the ER or golgi inactive enzymes are 'zymogens'
73
phosphorylation
co/ posttranslational mod occurs on hydroxyl groups of serine, threonine, tyrosine residues catalysed by kinases reversed by phosphatases
74
carbonate chains
many proteins secreted/ that are part of cell membrane can occur in golgi
GMC
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