LECTURE 14
RNA: TRANSCRIPTION & PROCESSING
chapter 8
 announcements
 key ideas
 RNA
 transcription in
Prokaryotes
 transcription in Eukaryotes
ANNOUNCEMENTS
missed or late for quiz ?
 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 …
 M 3:00 – 4:30 & F 11:00 – 12:30 in WHI 111
extra-credit tutoring … in progress, check web page
extra-credit seminar WHI AUD F 3:30
 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
CH9

CH7


CH8 KEY IDEAS
DNA transcription RNA translation protein
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
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
RNA
RNA
RNA
RNA
RNA
RNA
DNA
usually single-stranded,
complex 3D structures
 double-stranded,
antiparallel
ribose sugar
 deoxyribose
C2
C2
OH
uracil pyrimidine
U
A
H
 thymine
T
A
RNA
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
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
TRANSCRIPTION IN PROKARYOTES
DNA coding strand = nontemplate strand
 sequence homologous to transcribed
mRNA
 DNA has A, RNA has U
DNA template strand  mRNA
TRANSCRIPTION IN PROKARYOTES
genes transcribed from one but either DNA strand
transcription by complementary pairing of bases
catalyzed by RNA polymerase (RNA pol)
new mRNA grows 5'  3'
RNA pol moves along DNA template strand 3'  5'
5'
3'
5'
3'
TRANSCRIPTION IN PROKARYOTES
normally illustrate in other direction for
new
mRNA grows 5'  3'
convenience
RNA pol moves along DNA template strand 3'  5'
TRANSCRIPTION IN PROKARYOTES
transcription of rRNA genes in Triturus
TRANSCRIPTION IN PROKARYOTES
3 distinct stages of transcription
1. initiation
2. elongation
3. termination
TRANSCRIPTION IN PROKARYOTES
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
closed promoter complex
open promoter
complex
 different  factors recognize different DNA
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
3
'
elongation
new mRNA grows 5'  3'
RNA pol moves along
3
'
5
'
DNA template strand 3' 
5'
ribonucleoside
triphosphate added to 3'
end of nth base
5
'
TRANSCRIPTION IN PROKARYOTES
elongation
3
'
new mRNA grows 5'  3'
RNA pol moves along
DNA template strand 3' 
5'
pyrophosphate ion
released
3
'
5
'
5
'
TRANSCRIPTION IN PROKARYOTES
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'
TRANSCRIPTION IN PROKARYOTES
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
TRANSCRIPTION IN PROKARYOTES
termination
RNA pol recognizes signal for chain termination
2 mechanisms for termination in prokaryotes
1. direct termination - termination sequence
2. rho-dependent
TRANSCRIPTION IN PROKARYOTES
termination
rho-dependent termination
no U-residues formed
no hairpin loop
rho binds to rut site
on RNA
TRANSCRIPTION IN PROKARYOTES
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 PROKARYOTES
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 Eukaryotes
1. RNA synthesis
TRANSCRIPTION IN EUKARYOTES
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
TRANSCRIPTION IN EUKARYOTES
Prokaryotes:
 RNA pol only
Eukaryotes:
 RNA pol II +
general transcription
factors (GTFs) 
mRNA
DNA
GTFs
TRANSCRIPTION IN EUKARYOTES
differences because of complexity in Eukaryotes
1. RNA synthesis
2. RNA processing
TRANSCRIPTION IN EUKARYOTES
Prokaryotes:
 no processing
Eukaryotes:
 processed
before being
transported to the
cytoplasm
TRANSCRIPTION IN EUKARYOTES
differences because of complexity in Eukaryotes
1. RNA synthesis
2. RNA processing
3. chromosome organization
4. split genes
TRANSCRIPTION IN EUKARYOTES
Prokaryotes:
 naked DNA (nearly)
Eukaryotes:
 chromatin –
euchromatin &
heterochromatin
TRANSCRIPTION IN EUKARYOTES
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
°1 mRNA shortened
before transport to
cytoplasm
chicken ovalbumin
DNA/mRNA hybrid 
coding sequences...
exons
intervening sequences...
introns
EUKARYOTIC RNA
°1 mRNA shortened
before transport to
cytoplasm
chicken ovalbumin
DNA/mRNA hybrid 
coding sequences...
exons
intervening sequences...
introns
introns spliced from
TRANSCRIPTION IN EUKARYOTES
summary...
genomic DNA 
RNA exons + introns
transcribed  1°
mRNA transcript
processed  cap &
polyadenylation
spliced  splicing
intermediate
spliced  mature
mRNA
TRANSCRIPTION IN EUKARYOTES
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...
TRANSCRIPTION IN EUKARYOTES
1 gene  multiple functions by alternative splicing
e.g., -tropomyocin gene...
TRANSCRIPTION IN EUKARYOTES
gene splicing mechanism...
sequence homologies at exon-intron splice junctions
= consensus sequences for splicing enzymes...
TRANSCRIPTION IN EUKARYOTES
spliceosome
snRNA aligns sequences correctly for splicing
TRANSCRIPTION IN EUKARYOTES
splicing reaction
intron lariat structure
introns excised by 2
transesterification reactions
TRANSCRIPTION IN EUKARYOTES
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
TRANSCRIPTION IN EUKARYOTES
self splicing mechanisms, mRNA catalyzes reaction
(called ribozymes)
TRANSCRIPTION IN EUKARYOTES
self splicing mechanisms, mRNA catalyzes reaction
spliceosome-catalyzed mechanism
QUESTIONS – CHAPTER 8
TUTORIAL 6