Gene expression II: from RNA to protein Flashcards
what does RNA stand for?
Ribonucleic acid
what is RNA?
the product of transcription from a DNA template
how much more RNA in a cell than DNA?
10x more
what 3 general factors make DNA and RNA different?
- different chemical structure
- different physical structure
- different biological role
what’s different chemically about RNA to DNA? (2)
- ribose contains 2-OH, whereas deoxy contains 2-H (deOXY)
- the base uracil is used instead of thymine
how does the oxygenated ribose as opposed to deoxygenated DNA affect stability?
it’s less stable (RNA)
what’s the difference between thymine and uracil?
uracil doesn’t have a CH3 group that thymine does have
what are the biological roles of RNA?
- transfer of information from DNA to protein (mRNA)
- synthesis of proteins (rRNA and tRNA)
- processing of messenger RNA (snRNA)
- processing and modification of ribosomal RNA (snoRNA)
- catalytic RNA (self-splicing introns)
how much RNA does a single cell contain?
10pg/ 10^-11g
what proportion of RNA in cells is ribosomal?
80-85%
what proportion of RNA in cells is low molecular weight RNA e.g. snRNAs, tRNAs etc.?
10-15%
what proportion of RNA in cells is messenger?
1-5%
what are the physical properties of RNA?
- single stranded
- can base pair with itself to fold into complex structures which depends on its sequence
the regulation of the amount or useage of certain RNAs is important for their function, what is this regulation controlled by? (3)
- synthesis
- regulated degradation
- translation efficiency
when iron binds to transferrin what happens?
it’s transported into cells via a receptor
what is iron uptake regulated by?
degradation of transferrin receptor mRNA
what structures are found within the transferrin receptor mRNA?
AU rich loop
what does this Au rich loop act as?
binding site for IRE-BP (iron response element binding protein)
what 3 processes does mRNA go through to become mature?
capping
cleavage/polyadenylation
splicing
which end of RNA is modified by capping?
5’
what happens during capping?
the 5’ end of GTP molecule joins the 5’ end of the RNA and 1 phosphate is lost
methylation at the 2’ position of first 2 nucleotides and on the added G
what is the purpose of capping? (5)
- increases the stability of mRNA
- required for efficient splicing
- nuclear export (leaving the nucleus)
- translation initiation (translation can’t start without capping)
what does GTP stand for?
Guanosine TriPhosphate
how is RNA modified on the 3’ end?
3’ cleavage and polyadenylation
what signals cleavage/ polyadenylation? (2)
AAUAAA
G/U or just U rich sequence
what are the steps for 3’cleavage and polyadenylation?(3)
- cleavage and polyadenylation specificity factor (CPSF) binds to AAUAAA
- cleavage stimulatory factor (CstF) binds to G/U- recruits cleaving factors and poly-A polymerase (PAP)
- cleavage and addition of polyA
what is PolyA tailing linked to?(2)
termination of transcription
stability and translation efficiency
what signals are required for pre-mRNA splicing?
conserved sequences at the
5’ splice site, 3’ splice site
and branchpoint region
does splicing happen in 1 or several stages?
several
what do splicing sites (5’ donor site and 3’ acceptor site) do?
tell apparatus where to cut the RNA
what does the branch point contain?
an A in the sequence
how many steps in the splicing mechanism?
2
what happens in the first step of the splicing mechanism?
cleavage at the 5’ splice site and lariat formation at branchpoint sequence- RNA is cut after exon 1 and the GU of the sequence joins to the A in the branch point (the free 2’ Hydroxyl of A joins to the phosphate of GU)
what happens in the second step of the splicing mechanism?
cleavage at 3’ splice site. Removal of intron region and exon ligation (join up)- the introns are cut out and exons are kept
what carries out splicing?
small nuclear ribonucleoprotein particles (snRNPs/ snurps) and protein splicing factors
what makes snRNPs?
snRNA + small nuclear RNA
which snRNPs are involved in splicing?
U1, U2, U4, U5 and U6
which snRNPs form the spliceosome?
U2
U5
U6
What are the steps of the spliceosome assembly pathway? (5)
- exon 1 and intron boundry recognised by U1 and U2
- U4, U5 and U6 are recruited
- complex is called the spliceosome
- U4, U5 and U6 displace U1- still associated but is pushed away
- to join the 2 exons together, they must be brought close together and have intron RNA removed- which is done by U2,U5 and U6
what’s alternative splicing?
exon inclusion/inclusion to alter a protein sequence
give an example of alternative splicing use:
1 from:
- sex determination in fruit flies
- control of flowering time
- olfactory receptor diversity
what’s the purpose of alternative splicing?
to produce different proteins with different functions from a single gene
what’s the technical term for protein synthesis?
translation
during translation mRNA–>
protein
how many Amino acids are there?
20
how was the genetic code broken?
polynucleuotides e.g. UUUUUUUU were looked at to see which amino acids they made, then more varying polynucleotides were looked at e.g. UAUAUAU
do some AAs have more than one codon?
yes e.g. 6 different codons code for Leucine
what’s a reading frame?
the sequence of codons from a specific start codon to a specific stop codon
what codon does almost every protein chain initiate with?
AUG (methionine)
what are the 3 stop codons?
- UAA
- UGA
- UAG
are all methionine codons start codons?
no
what is the role of the 3’ end of tRNA?
acceptor arm, covalently bonds to AA
what is on the opposite side of tRNA to the AA?
anti-codon
what’s an anti-codon?
a codon which recognizes the codon sequences of the mRNA that’s the same as the AA on the tRNA acceptor arm (complementary)
what’s the function of tRNA?
to specifically link to a particular amino acid and to recognise a codon in mRNA (ensures amino acid-codon match)
what pairs tRNA to amino acids?
aminoacyl-tRNA synthetases
each synthetase is specific for …
amino acid and tRNA
can some tRNAs recognise more than one codon?
yes- some AAs have more than one codons
what does wobble allow?
unconventional base pairing between third base in codon and first base in anticodon - allows one tRNA to recognise more than one codon
what are the 3 stages of translation?
- initiation
- elongation
- termination
what are ribosomes?
organelles where protein synthesis/ translation takes place
made of RNA skeleton with sites where proteins can build
How many svedbergs in prokaryotic ribosome?
70S
what are the subunits of prokaryotic ribosome?
50S
30S
what are the subunits of ribsomes made of?
difference species of rRNA and proteins
how many svedbergs is a eukaryotic ribosome?
80S
what are the subunits of a eukaryotic ribosome?
60S
40S
what is a Svedberg?
a measure of the sedimentation rate of suspended particles centrifuged under standard conditions
S=size, not mass
what’s the 2D structure of rRNA like?
elaborate made up of 40 or more stem loops
what does the 3D structure of rRNA contain? (3)
ridges
crevices
holes
what’s the purpose of the holes in rRNA?
may be for interaction with mRNA or tRNAs to slot in with an AA
how are amino acids activated?
by ATP which esterifies it to form an AMP- amino acyl, AA
AA+ATP= AA-AMP (monophosphate) +PP(2 phosphates
How do amino acids join to tRNA?
the 3’OH group of tRNA acts as an acceptor arm for the activated AA, they join and AMP is released
(AA-AMP+ tRNA–> AA-tRNA+AMP)
what is an amino-acyl?
an AA joined to something else
is the shine-dalgarno sequence seen in prokayotes or eukaryotes?
prokaryotes
what is the shine-dalgarno sequence?
sequence which defines which is the correct initiation codon- not all methionines are equal (it needs the shine-dalgarno surrounding it)
what does the shine-dalgarno interact with?
the 16S rRNA of the 30S small subunit to identify the site of initiation of protein synthesis
what’s the shine- dalgarno sequence called in eukaryotes?
Kozak sequence
can initiation occur without methionine?
yes sometimes if the kozak/ shine-galdarno sequence is ‘good enough’
how many tRNAs are there for methionine?
2
what is each of the tRNAs for methionine used for?
one for initiation (tRNAi)
one for elongation
what’s different about prokaryotic methionine as opposed to eukaryotic?
it’s formylated
what happens in the process of initiation?
- IF2 engages with 30S subunit
- messenger RNA (mRNA) and 1st methionine join onto the 30S subunit- this forms the ternary complex (ribosome, mRNA and tRNA and IFs)
- the large subunit is assembled on the ternary complex- it has 2 slots (P and A)
- initiator methionine is lined up with the P slot
- IF2 and IF3 are ejected and GTP is hydrolysed to GDP and 2 ATPs are hydrolysed to 2 ADPs
- initiation complex has been formed
what are IFs?
initiation complexes
what does IF1 do?
blocks A site to tRNAi-met, inhibiting premature 30S-50s interaction
what does IF2 do?
tags tRNAi and regulates entry into ribosome
what does IF3 do?(3)
- inhibits premature 30S-50s interaction
- stabilises free 30S
- accuracy check for tRNAi-met binding
what happens during elongation?
- tRNA-meti is the only tRNA which can bind to the P site
- the next aminoacyl tRNA comes in and binds to the A site, brought there by EFTu, using energy form the hydrolysis of GTP to GDP
- AAs join together by peptide bonds and tRNA-meti is ejected from the complex- leaving an occupied A site, not P (as nothing else can bind to it)
- the ribsosome translocates (moves along) the mRNA so P site is lined up with next codon, where the next tRNA comes in and so on and so forth
what are EFs?
Elongation Factors
what are the 2 EFs?
- EF-Tu
- EF-G
what does EF-Tu do?
mediates aminoacyl-tRNA entry to the ribosome
what does EF-G do?
mediates translocation of ribosome
what happens at termination?
- RF-GTP binds to A site when the termination codon appears
- hydrolysis of polypeptide chain from tRNA (the polypeptide is ejected) by RF
- then there’s dissociation of the tRNA,RF and GTP
what’s an RF?
release factor
how many prokaryotic RFs are there?
3
what are the RFs?
RF1
RF2
RF3
What does RF1 do?
specific to UUA/UAG stop codons to cause termination when these are reached
what does RF2 do?
specific to UAA/UGA stop codons to cause termination when these are reached
what does RF3 do?
helps RF1/2 bind to ribosome
what does GTP stand for?
Guanosine Triphosphate
what does GTP do?
provides energy by being hydrolysed
what does GTP do in inititation?
Adds a large subunit
what does GTP do in elongation?
adds amino-acyl-tRNA with EF-Tu
peptide synthesis
transloacation
what does GTP do in termination?
releases the peptide chain and dissociates ribosome from complex
which RNA codes for protein?
mRNA
which RNA is involved in processing mRNA?
snRNA
which RNA is involved in siting and catalysis of protein synthesis?
rRNA
which RNA is involved in the processing of ribosomal RNA?
snoRNA
which bases can I (inosine) bond with?
C,A,U — not G
which base is inosine most similar to?
G
