Transcription 11/3/05Transcription 11/3/05
Stable RNAStable RNA rRNA -Structural component of rRNA -Structural component of
ribosomesribosomes tRNA-Adaptors, carry aa to ribosometRNA-Adaptors, carry aa to ribosome SynthesisSynthesis
Promoter and terminatorPromoter and terminator Post-transcriptional modification (RNA Post-transcriptional modification (RNA
processing)processing) EvidenceEvidence
Both have 5’ monophospatesBoth have 5’ monophospates Both much smaller than primary transcriptBoth much smaller than primary transcript tRNA has unusual bases. EX pseudouridinetRNA has unusual bases. EX pseudouridine
tRNA and tRNA and rRNA rRNA
ProcessingProcessing Both are Both are
excised from excised from large primary large primary transcriptstranscripts
11ºº transcript transcript may contain may contain several tRNA several tRNA molecules, molecules, tRNA and rRNAtRNA and rRNA
rRNAs simply rRNAs simply excised from excised from larger larger transcripttranscript
tRNAs modified tRNAs modified extensivelyextensively
5. Base modifications
Examples of Covalent Modification of Nucleotides in Examples of Covalent Modification of Nucleotides in tRNAtRNA
N
NN
N
NCH3H
6
N
NHN
N
O
N
NN
N
NCH2H
6
CH C
CH3
CH3
N
NHN
N NH2
CH3 O
7
N
NHHH
HH
O
O
HN
C
NH
O
O
12
34
56
N
NH
O
O
OH2C
CHO O
5
N
N
NH
O
CH33
N
NH3C
NH2
O
5
H
Base
H
CH2
O O
H HO
1'
2'
O
CH3
N6-Methyladenylate(m6A)
N6-Isopentenyladenylate(i6A)
Inosinate(I)
7-Methylguanylate(m7G)
Dihydrouridylate(D)
Pseudouridylate (Ψ)(ribose at C-5)
Uridylate 5-oxyacetic acid
(cmo5U)
3-Methylcytidylate(m3C)
5-Methylcytidylate(m5C)
2’-O-Methylated nucleotide(Nm)
Modifications are shown in blue.
Eukaryotic TranscriptionEukaryotic Transcription Regulation very complexRegulation very complex Three different pols Three different pols Distinguished by Distinguished by -amanitin -amanitin
sensitivitysensitivity Pol I—rRNA, least sensitivePol I—rRNA, least sensitive Pol II– mRNA, most sensitivePol II– mRNA, most sensitive Pol III– tRNA and 5R RNA moderately Pol III– tRNA and 5R RNA moderately
sensitivesensitive Each polymerase recognizes a Each polymerase recognizes a
distinct promoterdistinct promoter
Eukaryotic RNA Eukaryotic RNA Polymerases Polymerases
RNA Pol. Location Products -Amanitin Sensitivity
Promoter
I Nucleolus Large rRNAs (28S, 18S, 5.8S)
Insensitive bipartite promoter
II Nucleus Pre-mRNA, some snRNAs
Highly sensitive
Upstream
III Nucleus tRNA, small rRNA (5S),
snRNA
Intermediate sensitivity
Internal promoter and terminator
Eukaryotic Polymerase I Eukaryotic Polymerase I PromotersPromoters
RNA Polymerase IRNA Polymerase I Transcribes rRNATranscribes rRNA Sequence not well conservedSequence not well conserved
Two elementsTwo elements Core element- surrounds the Core element- surrounds the
transcription start site (-45 to + 20)transcription start site (-45 to + 20) Upstream control element- between -Upstream control element- between -
156 and -107 upstream156 and -107 upstream Spacing affects strength of transcriptionSpacing affects strength of transcription
Eukaryotic Polymerase II Eukaryotic Polymerase II PromotersPromoters
Much more complicatedMuch more complicated Two partsTwo parts
Core promoterCore promoter Upstream elementUpstream element
Core promoterCore promoter TATA box at ~-30 basesTATA box at ~-30 bases Initiator—on the transcription start siteInitiator—on the transcription start site Downstream element-further downstreamDownstream element-further downstream
Many natural promoters lack recognizable Many natural promoters lack recognizable versions of one or more of these sequencesversions of one or more of these sequences
TATA-less PromotersTATA-less Promoters
Some genes transcribed by RNA pol II lack Some genes transcribed by RNA pol II lack the TATA boxthe TATA box
Two types:Two types: Housekeeping genes ( expressed constitutively). Housekeeping genes ( expressed constitutively).
EX Nucleotide synthesis genesEX Nucleotide synthesis genes Developmentally regulated genes. EX Homeotic Developmentally regulated genes. EX Homeotic
genes that control fruit fly development. genes that control fruit fly development. Specialized (luxury) genes that encode cell-Specialized (luxury) genes that encode cell-
type specific proteins usually have a TATA-type specific proteins usually have a TATA-boxbox
mRNA Processing in mRNA Processing in EukaryotesEukaryotes
Primary transcript much larger than Primary transcript much larger than finished productfinished product
Precursor and partially processed Precursor and partially processed RNA called heterogeneous nuclear RNA called heterogeneous nuclear RNA (hnRNA)RNA (hnRNA)
Processing occurs in nucleus Processing occurs in nucleus
Capping Capping mRNAmRNA 5’ cap is a 5’ cap is a reversedreversed
guanosine residue guanosine residue so there is a 5’-5’ so there is a 5’-5’ linkage between the linkage between the cap and the first cap and the first sugar in the mRNA.sugar in the mRNA.
Guanosine cap is Guanosine cap is methylated.methylated.
First and second First and second nucleosides in nucleosides in mRNA may be mRNA may be methylatedmethylated BAC
K
PolyadenylationPolyadenylation Polyadenylation occurs on the 3’ end of Polyadenylation occurs on the 3’ end of
virtually all eukaryotic mRNAs.virtually all eukaryotic mRNAs. Occurs after cappingOccurs after capping Catalyzed by polyadenylate polymeraseCatalyzed by polyadenylate polymerase Polyadenylation associated with mRNA Polyadenylation associated with mRNA
half-lifehalf-life Histones not polyadenylatedHistones not polyadenylated
Introns and Exons Introns and Exons Introns--Introns--
Untranslated Untranslated intervening intervening sequences in mRNAsequences in mRNA
Exons– Translated Exons– Translated sequencessequences
Process-RNA Process-RNA splicingsplicing
Heterogeneous Heterogeneous nuclear RNA nuclear RNA (hnRNA)-Transcript (hnRNA)-Transcript before splicing is before splicing is complete complete
Splicing OverviewSplicing Overview Occurs in the nucleusOccurs in the nucleus hnRNAs complexed with specific proteins, hnRNAs complexed with specific proteins,
form a form a ribonucleoprotein particle (RNP)ribonucleoprotein particle (RNP) Primary transcripts assembled into Primary transcripts assembled into hnRNPhnRNP Splicing occurs on Splicing occurs on spliceosomes spliceosomes consist ofconsist of Small nuclear ribonucleoproteins Small nuclear ribonucleoproteins (SnRNPs)(SnRNPs)
components of spliceosomescomponents of spliceosomes Contain small nuclear RNA (Contain small nuclear RNA (snRNAsnRNA)) Many types of snRNA with different functions in Many types of snRNA with different functions in
the splicing processthe splicing process
Splice Site RecognitionSplice Site Recognition
IntronsIntrons contain invariant 5’-GU and 3’-AG contain invariant 5’-GU and 3’-AG sequences at their borders (GU-AG Rule)sequences at their borders (GU-AG Rule)
Internal intron sequences are highly variable Internal intron sequences are highly variable even between closely related homologous even between closely related homologous genes.genes.
Alternative splicing allows different proteins Alternative splicing allows different proteins from a single original transcriptfrom a single original transcript
Simplified Splicing Simplified Splicing MechanismMechanism
RNA pol III RNA pol III Precursors to tRNAs,5SrRNA, other small Precursors to tRNAs,5SrRNA, other small
RNAsRNAs Promoter Type IPromoter Type I
Lies completely within the transcribed regionLies completely within the transcribed region 5SrRNA promoter split into 3 parts5SrRNA promoter split into 3 parts tRNA promoters split into two partstRNA promoters split into two parts
Polymerase II-like promotersPolymerase II-like promoters EX. snRNAEX. snRNA Lack internal promoterLack internal promoter Resembles pol II promoter in both sequence Resembles pol II promoter in both sequence
and positionand position
DNAse DNAse FootprintingFootprinting
Use: promoter Use: promoter IDID
End Label End Label template strandtemplate strand
Add DNA binding Add DNA binding proteinprotein
Digest with Digest with DNAse IDNAse I
Remove proteinRemove protein Separate on gelSeparate on gelProtected
region