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