lecture 14 rna: transcription & processing

LECTURE 14 RNA: TRANSCRIPTION & PROCESSING

chapter 8 announcements key ideas RNA transcription in Prokaryotes transcription in Eukaryotes

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same documentation required as for exams no makeup… average of those you write

exam 2 … M 11.13 in class, as listed on calendar extra tutorial help …

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Dr. Juli Wade, Michigan State U. Hormonal and Genetic Influences on Sexual

Differentiation of the Zebra Finch Song System

CH8 KEY IDEAS 3 processes of information transfer in genetics

CH8

CH7

CH9

CH8 KEY IDEAS

translation requires transfer RNAs & ribosomes information transfer by non-overlapping triplet code special DNA sequences signal initiation & termination

of transcription & translation in Eukaryotes only... mRNA transcripts are processed prior to translation noncoding introns interrupt coding exon sequences introns are spliced out of 1° mRNA final mRNA

DNA transcription RNA translation protein

CH8 KEY IDEAS transcription & translation

RNA DNA protein... RNA intermediate? pulse-chase experiments with RNA precursors... pulse

radioactive, chase nonradioactive, autoradiograph

synthesis of RNA in nucleus protein in cytoplasm

usually single-stranded, double-stranded, complex 3D structures antiparallel

ribose sugar deoxyribose C2

H

C2

OH

RNA

AU AT

RNA DNA

uracil pyrimidine thymine

classes

1. informational mRNA: translated polypeptides

2. functional tRNA: bind & transport amino acids rRNA: components of ribosomes snRNA: participate in modifying rRNA

components of spliceosomes

RNA

TRANSCRIPTION IN PROKARYOTES

T2 bacteriophage infection of E. coli pulse-chase with radiolabeled uracil (RNA-precursor) labeled RNA recovered only immediately after pulse...

rapid turnover phage-induced

RNA similar to T2 DNA... DNA RNA

TRANSCRIPTION IN PROKARYOTES in vitro synthesis shows RNA DNA...

DNA template & complementary RNA bases

similar to DNA replication, with DNA polymerase... transcription enzyme... RNA

polymerase ?

TRANSCRIPTION RNA synthesized from 1

or both DNA strands? hybridization experiment complementary DNA

strands have different densities

each RNA hybridizes to only 1 DNA strand

transcription is asymmetrical

TRANSCRIPTION IN PROKARYOTES DNA template for mRNA transcription

note directional nature of event


DNA coding strand = nontemplate strand sequence homologous to transcribed mRNA DNA has A, RNA has U

DNA template strand mRNA


new mRNA grows 5' 3' RNA pol moves along DNA template strand 3' 5'

5'3'

5'3'

error!error!error!

genes transcribed from one but either DNA strand transcription by complementary pairing of bases catalyzed by RNA polymerase (RNA pol)


RNA pol moves along DNA template strand 3' 5'

new mRNA grows 5' 3' normally illustrate in other direction for convenience


transcription of rRNA genes in Triturus nucleus


3 distinct stages of transcription

1. initiation

2. elongation

3. termination


initiation

2 types of sequences in prokaryotic genes

1. promoter sequences signal initiation

2. coding sequences are transcribed

TRANSCRIPTION IN PROKARYOTES initiation 2 regions of homology among promoter sequences

consensus sequences = RNA pol binding sites

TRANSCRIPTION IN PROKARYOTES initiation factor binds to –10 and –35 consensus regions initiates melting or denaturing of DNA transcription begins when subunit dissociates

open promoter complex

different factors recognize different DNA sequences

closed promoter complex

TRANSCRIPTION IN PROKARYOTES elongation new mRNA grows 5' 3' RNA pol and transcription bubble moves 3' 5'

along the DNA template strand

5'3'

TRANSCRIPTION

elongation

new mRNA grows 5' 3'

RNA pol moves along DNA

template strand 3' 5'

ribonucleoside triphosphate

added to 3' end of nth base

3'

5'

5'

3'

elongation

new mRNA grows 5' 3'

RNA pol moves along DNA

template strand 3' 5'

pyrophosphate ion

released

3'

3'

5'

5'


termination RNA pol recognizes signal for chain termination 2 mechanisms for termination in prokaryotes

1. direct termination - termination sequence


mRNA 3' UTR DNA CG rich + AAA...(6+)

5'3'

termination

direct termination, RNA pol recognizes ~40 bp terminator sequence on template

RNA forms hairpin loop

poly-A tail bonds weak

signal to release RNA pol

ATP-independent


termination

RNA pol recognizes signal for chain termination

2 mechanisms for termination in prokaryotes

1. direct termination - termination sequence

2. rho-dependent


termination

rho-dependent termination

no U-residues formed

no hairpin loop

rho binds to rut site

on RNA



termination

rho-dependent termination

no U-residues formed

no hairpin loop

rho binds to rut site

on RNA

rho “pulls” RNA from

RNA pol

TRANSCRIPTION IN EUKARYOTES

differences because of complexity in Eukaryotes1. RNA synthesis


Prokaryotes:

RNA pol all types of RNA

polycistronic mRNA

Eukaryotes:

RNA pol I rRNA (except 5S rRNA)

RNA pol II monocistronic mRNA, some snRNA

RNA pol III tRNA, 5S rRNA, some snRNA


Prokaryotes:

RNA pol only

Eukaryotes:

RNA pol II + general transcription factors (GTFs) mRNA

DNA GTFs


differences because of complexity in Eukaryotes1. RNA synthesis2. RNA processing


Prokaryotes:

no processing

Eukaryotes:

processed before being transported to the cytoplasm


differences because of complexity in Eukaryotes1. RNA synthesis2. RNA processing3. chromosome organization4. split genes


Prokaryotes:

naked DNA (nearly)

Eukaryotes:

chromatin – euchromatin & heterochromatin


3 distinct stages of transcription

1. initiation

2. elongation

3. termination

TRANSCRIPTION IN EUKARYOTES initiation GTFs TATA sequence

before RNA pol II binding attract RNA pol II positions complex

GTFs added after preinitiation complex transcription bubble

TRANSCRIPTION IN EUKARYOTES elongation GTFs added after

preinitiation complex transcription bubble RNA pol II carboxyl

tail domain (CTD) phosphorylated...

TRANSCRIPTION IN EUKARYOTES 2 types of processing

1. cotranscriptional

TRANSCRIPTION IN EUKARYOTES cotranscriptional processing – CTD dependend

guanyltransferase adds 7'-methylguanosine “cap” to 5' end of mRNA

protects single stranded RNA from degradation

TRANSCRIPTION IN EUKARYOTES cotranscriptional processing – CTD dependend

splicing by spliceosomes (... stay tuned)

TRANSCRIPTION IN EUKARYOTES termination

AAUAAA sequence near 3' end initiates cleavage

... by endonuclease ~ 20 bp downstream

TRANSCRIPTION IN EUKARYOTES 2 types of processing

1. cotranscriptional

2. posttranscriptional

TRANSCRIPTION IN EUKARYOTES posttranscriptional processing

poly(A) polymerase adds poly(A) tail of 150-200 adenosine residues to 3' end cleavage site

complete °1 mRNA

EUKARYOTIC RNA

°1 mRNA shortened before transport to cytoplasm

chicken ovalbumin DNA/mRNA hybrid

coding sequences... exons

EUKARYOTIC RNA



coding sequences... exons intervening sequences...

introns

EUKARYOTIC RNA



coding sequences... exons intervening sequences...

introns introns spliced from mRNA genomic DNA RNA

exons + introns

summary... genomic DNA

RNA exons + introns transcribed 1°

mRNA transcript processed cap &

polyadenylation spliced splicing

intermediate spliced mature

mRNA


1 gene multiple functions by alternative splicing

multiple gene functions in different... tissues developmental stages

different mRNAs from same 1° mRNA transcript... e.g., -tropomyocin gene...


1 gene multiple functions by alternative splicing e.g., -tropomyocin gene...


gene splicing mechanism... sequence homologies at exon-intron splice junctions = consensus sequences for splicing enzymes...


spliceosome snRNA aligns sequences correctly for splicing


splicing reaction intron lariat structure introns excised by 2

transesterification reactions


splicing reaction intron lariat structure introns excised by 2

transesterification reactions snRNA + proteins small

ribonuclear particles (snRNPs) snRNP + 1° mRNA transcript

spliceosome spliceosome catalyzes splicing

transesterification reactions


self splicing mechanisms, mRNA catalyzes reaction (called ribozymes)


self splicing mechanisms, mRNA catalyzes reaction spliceosome-catalyzed mechanism


TUTORIAL 6

QUESTIONS – CHAPTER 8

lecture 14 rna: transcription & processing

Documents

dna polymerase

rna precursors

autoradiographsynthesis

rna intermediate

dna strandtranscription

t2 dna

dna rnatranscription

dna replication