Rna Stuff

Front 1

RNA transcription basics and bact diff

Back 1

-transc.=DNA to RNA
-transl.=RNA to Prot
-make RNA 5' to 3'
-RNA synth does NOT require a primer
-promotor=seq in DNA tells RNA where to start
Diff in bact:
-template in bact is DNA, chromatin in animals
-1 RNA pol, binds directly to promotor
-E coli promotor=AT rich at -10, TTGA at -35
-in animal RNA must transport out of nuc

Front 2

RNA types

Back 2

-mRNA-10,000-20,000 different-encode info to synth prots
-rRNA-28S and 18S
-tRNA-small 70-80NTs, carry AA's

Front 3

RNA polymerases

Back 3

-each composed of 2 large subunits and several smaller prots
-each recognizes specific set of promotors
-RNA pol don't proofread
-RNA pol I=40% of synth
-RNA pol II=50% of synth
-RNA pol III=10% of synth

Front 4

RNA pol II

Back 4

-Synth RNA from all genes encoding prots
-***TFIID and TBP are required for transc. by RNA pol II even w/o TATA box
PROMOTORS: TATA box~-20-25(genes expressed at hight rate), G-C rich promotor(about 100NTs long) upstream of start site (genes exp. at low rate=no tata box)
PROCESS:
-TFIID contains TBP(TATA-binding prot), TBP binds the TATA box-protecting from histones (TBP binds to minor groove, bending DNA)
-Other prots in TFIID=TAF's(TBP associated factors)-recruit RNA pol II
-RNA pol II starts transc. and leaves TFIID at promotor site to recruit more

Front 5

RNA pol I

Back 5

-transcribes gene for 18S and 28S rRNA
-Uses TBP and TAF also, though they don't specifically bind DNA b/c no TATA box
PROMOTOR:specific sequence 5' of transc. site(core element), and similar seq 100NTs 5' of start site(upstream core element)
PROCEDURE:
-UBF(upstream binding factor)-1 molec binds core elem. and 1 molec binds up. core elem. -protect from histones
-UBF binds SL1(composed of TBP and TAF's
-SL1 recruits RNA pol I
-pol I starts transc. and leaves factors behind to recruit.

Front 6

RNA Pol III

Back 6

-synth many small RNA's (tRNAs, some nuclear RNA's, and 5S rRNA)
PROMOTORS:
A)tRNA gene has a and b boxes in coding part of gene(conserved and important for structure)
B)5S-rRNA gene has c box in coding part
PROCESS:
A)tRNA:
-TFIIIC binds to a and b boxes
-TFIIIC recruits TFIIIB to region near 5' end of gene
-TFIIIB composed of TBP and two other TAF's, recruits pol III and initiates transc.
B)5S-rRNA:
-TFIIIA binds c-box
-TFIIIA recruits TFIIIC

Front 7

RNA pol II continued

Back 7

-make pre-mRNA (introns and exons)
CAP:
-Add 5' "cap" to pre-mRNA as it comes out back of RNA pol II~20-30NTs long
-Cap 7-CH3-guanosine that makes 5' end look like 3', it protects mRNA from degredation
POLY A TAIL:
-added in conjunction w/ cleavage rxn to 3' end
-not encoded in DNA, added postransctiptionally
-need 2 cis elements:
1)AAUAAA-polyadenylation signal, 15-30NTs 5' of cleav. site, cleav. occurs after a CA or UA following AAUAAA seq
2)GU-rich seq-20-50NTs 3' of cleav seq

Front 8

pre mRNA splicing

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-Spliceosome-maching that removes introns, made of small nuc RNA's(snRNA's)
-exons-<300NTs each
CONSENSUS SEQ:
-donor site-first two NTs of intron(5' end) are alway GU
-Acceptor site-last two Nts of intron(3'end) are always AG
-Branch point-always A (uacuaAc)
PROCESS:
1)cleave at end of exon just befor GU-phosphate on GU and OH on 3' end of exon, GU is covalently joined to 2' OH of branch point (A~25-40NTs away)
***Concerted rxn-no E change, break 1 phosphodiester bond and form one
-forms "lariat intermediate"
2)break bond after AG, then join two exons
-also concerted rxn
-intron released as a circle w/ short tail

Front 9

trans-acting factors in splicing

Back 9

-U(1,2,4,5,6)snRNP/A, U2aF
A)-Initiate formation of spliceosome:U1 binds donor site (GU),U2 binds branch point, U2aF binds polypyrimidine tract and acceptor site(AG)
B)-bind tri-snRNP (U4,U5,U6), displaces U1, U5 binds both sites, U4 is keeping U6 inactive
C)U4 leaves, U6 catalyzes splicing while U5 holds exons to be put together
***-finished mRNA then exits to the cytoplasm

Front 10

rhibosomes and elongation of prot

Back 10

tRNAs w/ AA bound to elongation factor 1(EF1) for ribo to grab them from
PREP:
1)need 5' cap, poly A tail, and prots(tRNA-meti) for initiation
2)cell identifies 5' cap and scans for first AUG-give reading frame
START:
1)tRNA(1) in P-site w/ growing polypeptide chain
2)proper tRNA(2) comes and enters A-site, GTP->GDP locks tRNA(2) in place
3)Ribo catalyzes formation of peptide bond transfering peptide chain to tRNA(2) in A site
TRANSLOCATION:
-orig tRNA(1) leaves P-site and tRNA(2) is transfered to P-site(using E from GTP and catal. by elongation factor G)
TERMINATION:
-stop codons (UAA, UAG, UGA) recruit prot, not a tRNA
-prot binds in A site
-polypep chain transfered from tRNA in P-site to water and released

Front 11

ribo parts from rRNA

Back 11

-ribo consists of 2 subunits, each of which contain single large RNA molec
1)Large subunit(60S)-28S rRNA(5000Nts), 5S rRNA(120NTs), 5.8S rRNA(160Nts), 50 prots
2)small subunit(40S)-18S rRNA(2000Nts), 30 prots
**-18S, 5.8S, and 28S cleaved from larger precursor (RNA pol I)
**-5S made by RNA pol III

Front 12

ribo synth and processing

Back 12

-synth and processing occurs in nucleolus
-RNA pol I-makes 45SrRNA precursor(5' to 3':18S,5.8S,28S)
-45SrRNA is cleaved and assembled into ribo subunits and exported to cyto
-cleavage and modification requires prots and small nucleolar RNA's (snoRNA's)

Front 13

alternative splicing

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2 types:
1)more than one protein is produced from a single mRNA in a single cell
2)particular pre-mRNA is spliced differently in one cell type than another
**results from recognition or failure to recognize particular exons usually due to prots.

Front 14

splicing mutations

Back 14

mutation at 5' donor site-deletion of exon, exon is skipped b/c cell recognizes the end of exon,but it's covered by intron
E1-I1-E2-I2-E3-I3-E4 with a mutation on donor site of I2 results in:
E1-E3-E4
(Maybe acceptor site is same)