Transcription in Eukaryotes pages 10861097

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Transcription in Eukaryotespages 1086-1097
Different from Prokaryotes

• Eukaryotes have chromatin
• - transcription machinery must work around
  nucleosomes
• Eukaryotes have a nucleus
• - no coupled Transcription/Translation
• Eukaryotes have three different RNA Polymerases
• - Polymerase I ? large ribosomal rRNAs
• - Polymerase II ? mRNA (and many snRNPs)
• - Polymerase III ? tRNA 5S rRNA.
• Eukaryotes have more complex processing of mRNAs
• - 5 cap
• - 3 poly A tail
• - splicing

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Types of cellular RNAs

• rRNA
• 4 Ribosomal RNA components are part of ribosomes
• ribosomes have 2 subunits (40S and 60S)(S refers
  to their size)
• tRNA
• Transfer RNA is used during the synthesis of
  proteins- carries amino acids that are to be
  added to a growing polypeptide chain
• mRNA
• mRNA is translated into proteins
• snRNPs (small nuclear ribonucleoprotein
  particles)
• contain small RNAs that have structural or
  catalytic roles

3
Eukaryotic RNA Polymerases
- eukaryotic RNA polymerases are exceedingly
complex (in terms of subunit structure) more
than 10 subunits each (E. coli has 5) - they
exhibit variable sensitivity to ?-amanitin ( a
poison from a toadstool)
Pol I Insensitive to ?-amanitin ? rRNA
unaffected Pol II VERY sensitive ? No mRNA
produced Pol III Moderately sensitive ? less
tRNA made ? Drug sensitivity can be used to
determine which RNA polymerase synthesizes a
given RNA in vivo
- eukaryotic polymerases also exhibit a
sensitivity to the drug actinomycin D
4
Chemical Inhibitors of RNA polymerases
a) Rifampicin Inhibits initiation in prokaryotes
by binding to RNA polymerase.
b) Actinomycin D Inhibits transcription
elongation by all RNA polymerases by binding to
DNA and blocking the movement of the enzyme along
template
c) ?-Amanitin Inhibits elongation in Pol II gt
Pol III (it binds preferentially to Pol II)
5

• Eukaryotes also have one or two other types of
  RNA polymerases
• Mitochondrial
• Chloroplast
• - both of these are very similar to their
  bacterial counterparts

Euk. RNA polymerases bind DNA promoters with the
help of other proteins called transcription
factors (TFs) TFs bind DNA, then help RNA
polymerase to bind (they stabilize binding, e.g.
like CRP in prokaryotes)
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RNA Polymerase I
- found in the nucleolus, only used to make rRNA
(need to make 10,000,000 ribosomes in each cell
generation) - all three rRNAs transcribed as a
single 45S precursor RNA (13,000 nt
long) -Precursor is cleaved during ribosome
assembly (also in nucleolus) (this gene structure
ensures that the subunits are always made in
equal numbers)
18S
40S
Small ribosome subunit
45S
proteins
5.8S
60S
Large ribosome subunit
5S (from nucleus)
28S
7
- rRNA genes are repeated in tandem arrays in
most eukaryotes 100-500 copies / cell (humans
have 200/haploid genome) E. coli has 7 rRNA
genes -genes are arranged in clusters (humans
have 5) RNA polymerase I requires 2 TFs for
initiation TFIB Binds promoter TFIS
Stabilizes the binding of TFIB allows binding
of the RNA Pol I
8
DNA
pre-RNA
Figure 28.20
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RNA Polymerase III

• - synthesizes 5S tRNAs
• - three Transcription Factors involved
• TFIIIA only required for 5S transcription
• TFIIIB required for 5S tRNA transcription
• TFIIIC required for 5S tRNA transcription
• Binding order TFIIIA ? TFIIIC ? TFIIIB ? Pol III
• - transcribes genes that share features- they
  are small do not encode proteins and have
  unique regulatory sequences

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RNA pol III

• 5S rRNA gene
• First, TFIIIA binds to a 40 bp contact region
• This then facilitates the binding of TFIIIC/B and
  pol III
• 5S also complexes with TFIIIA-
• Leads to its inhibition when there is enough 5S
  in the cell (blocks its DNA binding)-feedback
  control loop
• tRNA genes
• RNAs that associate with amino acids
• Need to be modified from nascent state

11
The promoters have an unusual / surprising
structure (they are internal i.e. within the
coding regions)
47
96
1
TFIIIA
5S RNA
10
60
1
TFIIIC
t-RNA
12
- 5S rRNA genes present in 500-20,000 copies /
cell (reflects high demand for this RNA to form
ribosomes) - Termination of Pol III
transcription occurs at runs of UUUs - All
t-RNAs and some 5S RNAs need processing
3
5
Mature t-RNA
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Processing of tRNA

• 5 ends are cleaved by RNase P
• RNAse P an RNP complex contains RNA
  Protein. It is the RNA component of RNase P that
  is catalytic
• ? it is therefore a RIBOZYME
• 3 ends are cleaved by another nuclease
• Some of the bases are modified to some unusual
  forms
• Some t-RNAs need introns removed

3
5
RNAse P
intron
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Eukaryotic mRNA Transcription (RNA Polymerase II)

• In metazoans, mRNA is the key to cell
  specialization / adaptation ? complex regulatory
  mechanisms fine tuning of expression.
• - Amount level of expression
• - Timing temporal expression pattern
• Location spatial expression pattern
• Also there is often a need for co-ordinate
  regulation of unlinked genes
• There are (unlike t-RNAs or rRNAs) a much larger
  range of different mRNAs in a cell
• - all cellular proteins are derived from mRNAs

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• Difficult to purify any one out of the mixture
• Special cases
• Virus infected cells often produce large
  quantities of viral mRNA
• Specialized cell types produce special mRNA in
  large quantities.
• Red blood cells GLOBINS
• Silk-worm silk glands FIBROIN
• Most early research was done on viral mRNA
  labeled in vivo with 32P
• - 32P incorporates into rapidly synthesized
  material
• - since the virus has hijacked the cells
  metabolism, most of the new mRNAs are viral

one can of course reverse transcribe and clone
cDNAs from a pool of mRNAs
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mRNAs are transcribed as hnRNA (heterogenous
nuclear RNA) ? unprocessed form of the mRNA,
straight off of the gene template
3 tailing
5 capping
hnRNA
mRNA
Gene ?
Splicing
Transcriptional Regulation Gene transcribed as a
monocistronic message (One promoter ? one gene)
there are a few exceptions but polycistronic
messages are not typical
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• Pol II-mediated transcription is normally
  regulated by two control regions
• Promoter Proximal Region
• Enhancer Elements distant DNA sequences from
  the promoter gene
• Promoter Proximal regions
• - usually contains a TATA Box _at_ -25 position.
• Consensus TATAA/TA
• - sometimes TATA box is absent in genes that are
  expressed at low levels
• - TATA Box Bound by TFIID

18
Other elements are also present in proximal
region of the region A CCAAT box or GGGCG
(within 100-200 bp of transcription start site)
in 10-15 of genes Multiple TFs bind to promoter
region - ALL affect levels of gene expression -
some TFs only present in particular cells,
particular times, or both
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Figure 28.24

• there is an ordered assembly of transcription
  factors that bind the promoter before RNA pol II
  can bind and transcription can take place
• the combination of all of these polypeptides is
  referred to as the minimal pre-initiation
  complex

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2. Enhancers or Enhancer Elements
- up to 50,000 base pairs away - also bind
multiple regulatory proteins Enhancers are -
DNA elements that can operate thousands of bp
away from the promoter - can operate in either
orientation, on either strand
GENE
Promoter
21
Various Looping structures can bring the
enhancers with their bound regulatory proteins in
closer proximity to the promoter and minimal
pre-initiation complex
Figure 28.27
22
Enhancers (enhancer-binding factors) regulate
(activate or suppress) transcription from a
promoter at the normal RNA start site. -
individual enhancers can activate transcription
100x or more

• How can we study or detect enhancer function?
• Attach promoter sequence to a reporter gene
  (lac Z or GFP, Green Fluorescent Protein)
• Add enhancer sequence or suspected enhancer
  sequence
• Reintroduce DNA constructs into cells
• Assay the level of activity of reporter gene

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suspected enhancer sequence
Promoter
distance can be changed (also orientation)
Reporter Gene
Eukaryotic Cell
Detect activity (compare to promoter alone)
24
Different Types of pol II Control Elements Bind
Transcription Factors That Are Important for
Eukaryotic Gene Regulation
Different target genes can be co-ordinately
regulated in response to specific signals, if
their regulatory regions contain such consensus
elements
25

• There is a huge variety of enhancer and promoter
  binding proteins, although there are a few
  recurring strategies for sequence specific DNA
  binding
• Zinc Finger Proteins.
• Leucine Zippers.
• Helix-Turn-Helix Motifs
• No class is specific for a particular function,
  and examples from one type have a variety of roles

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Common DNA binding motifs in proteins
Figure 28.23
Zinc Fingers
Leucine Zipper
Helix-Turn-Helix
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• Zinc Fingers
• Example includes TFIIIA which binds to the
  promoter of the 5S gene
• - 9 tandemly repeated domains of 30 aa each
  containing 2 His and 2 Cys which binds one zinc
  (each domain is a zinc finger)
• - Each zinc finger can recognize a 3 bp sequence
• - 9 repeats may allow TFIIIA to partially release
  while pol III goes by.
• Schematic of a Zinc Finger, The Zn2 cation is
  held in place by two histidines on the
  alpha-helix and two cysteines on the beta-sheet
  motif

Figure 28.22
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Leucine Zipper
- protein dimer with each protein interacting
with the major groove of DNA and the other
protein subunit - in some cases DNA affinity is
regulated by modulating the dimerization of the
proteins
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mRNA Capping
A specialized structure (cap) at the 5-end of
mRNA. - added just after initiation of
transcript - Only added to RNA Pol II (mRNA)
transcripts - 5-cap is important for initiation
of protein synthesis Serves to position the mRNA
on the ribosome for translation - may stabilize
mRNA (protects 5end from nucleases)
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Figure 28.30 5-Cap on the end of mRNA
31
Poly A Tailing (Polyadenylation)

• When transcript passes a site with AAUAAA ( and
  20 more nucleotides beyond it required )
• A nuclease cleaves transcript 10 30 bases down
  from AAUAAA
• the AAUAAA sequence is the
  polyadenylation site
• Poly A polymerase adds 100 250 bases of poly A
• - Yeast 100 bases
• - Flies 150 bases
• - Mammals 250 bases

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PolyadenylationThe function of polyadenylation
is not completely defined- the presence of
polyA tail may relate to message stability, since
mRNAs without tails are more short-lived in
nucleus
Figure 28.29
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Poly A chain is nibbled in cytoplasm gt
Final range of Poly A size is 30 250 bases
(Poly A may stabilize mRNA) - cleavage at Poly
A site (AAUAAA) is required for transcript
termination. (RNA Pol II carries on
transcribing, fruitlessly, well past this site.
This garbage RNA is probably degraded, because
it lacks cap) - only Pol II transcripts (i.e.
mRNA) are polyadenylated (Histone mRNAs the
exception not polyadenylated) - AAUAAA nuclease
may be associated with RNA Pol II
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PolyA tail allows lab purification of mRNAs
away from other RNAs - by hybridizing to PolydT
chains attached to columns
RNA Mixture rRNAs, tRNAs, mRNAs
dT
dT
dT
dT
dT
dT
rRNA, tRNA
mRNA
5
35

• Splicing was first detected by R-Looping
• - take DNA from a gene mRNA coded by that gene
  make an RNA DNA hybrid.
• - examine in electron microscope
• Nowadays, this can be routinely detected by
  sequencing both genomic DNA cDNA (made from
  reverse transcribing mRNA)

DNA-RNA Hybrid
ssDNA Loop