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TRANSCRIPTION
1) No free 3'OH end needed for initiation.
2) No primer needed.
3) 5'
3' synthesis directionality.
4) DNA-dependent RNA polymerase copies from one
single-stranded region of DNA (antisense strand).
5) Ribonucleoside triphosphate precursors.
6) DNA-RNA duplex less stable than DNA-DNA duplex.
RNA falls away from transcription site.
Coding or Sense Strand
________________________________________
3'
5'
Has same sequence as mRNA transcript
(except that is possesses T instead of U)
DNA
Template or Antisense Strand
________________________________________
3'
5'
Directs synthesis of complementary mRNA transcript
transcription
mRNA 5' ________________________________________ 3'
Triphosphate precursors
Transcription
(ribonucleoside triphosphate precursors; rNTPs )
2P
UTP
UMP
2P
CTP
CMP
2P
GTP
GMP
2P
ATP
AMP
Differences in Gene Expression Between Prokaryotes and Eukaryotes
Prokaryotes
Eukaryotes
1.Unwinding by gyrases
and helicases, but no
single strand binding proteins.
1. Possible unwinding by
gyrases and helicases, but no
single strand binding proteins.
2. All RNA species are
synthesized by a single
RNA polymerase.
2. Three different RNA
polymerases are responsible
for the different classes
of RNA molecules.
3. mRNA is translated
during transcription
(coupled).
3. Translation is independent
from transcription.
Prokaryotes
Eukaryotes
4. First nucleotide is a
purine.
4. mRNA is processed before
transport to the cytoplasm,
where it is translated.
Caps and tails are added,
and internal portions of
transcript are removed
(splicing).
5. Genes are contiguous
segments of DNA that
are colinear with the
mRNA that is
translated into a
protein.
5. Genes are often split.
They are not contiguous
segments of coding sequences
rather, the coding sequences
are interrupted by intervening
sequences (introns).
6. mRNAs are often
polycistronic.
6. mRNAs are often monocistronic.
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A. Prokaryotic – Ex. lac operon model.
I = Repressor gene
lac = lactose
P = Promoter
O = Operator
3’
RNA polymerase (Holoenzyme) =
core unit + σ (sigma unit) =
5’
σ
termination
σ70 = vegetative, for
all RNAs
σ60 = nitrogen
metabolism
σ 32 = heat shock proteins
Initiation:
a) Holoenzyme binds to promoter site, as specified by
sigma subunit and unwinds short stretch of DNA.
b) Sigma subunit released and transcription begins past
promoter site.
Termination:
a) rho-independent: rich G-C dyad region followed by
series of A’s and U’s (6-8) on RNA transcript causes
formation of hairpin structure and consequently causes
RNA polymerase to be knocked off.
5'
b)
rho-dependent: rho protein (hexamer) binds with RNA transcript at
rut site and causes it to fold around it, and upon transclocation and
further wrapping pulls the transcript away from the polymerase.
B.
EukaryoticDifferent RNA polymerases, with no sigma units:
RNA polymerase I - rRNA
RNA polymerase II - pre-mRNA
RNA polymerase III - tRNA and other small
structural RNAs
Eukaryotic RNA Polymerase
RNA polymerase I (inside nucleolus) - makes structural RNA
(i.e., rRNA).
RNA polymerase II (in nucleoplasm) - makes heterogenous
nuclear RNAs or hnRNAs (i.e., precursor mRNAs or premRNAs).
RNA polymerase III (in nucleoplasm) - makes small adaptor
RNAs (i.e., small nuclear RNAs and tRNAs).
Only one RNA polymerase in prokaryotes. However, its
activity is much like that of RNA polymerase II in eukaryotes.
Initiation:
interact
(binding)
Trans-acting regulatory
proteins
1) Transcription Factors (TFs)
function in the positive
regulation of transcription but
are not part of RNA polymerase
(RNA Pol). TFs contain
two functional domains: i) DNA binding domain - binds selectively
to Promoters and Enhancers.
ii) trans- acting domain - involved
in protein - protein interactions
between TFs and between TFs and
RNA Pol in the Promoter.
See Fig. 1.
3'
Fig. 1
Cis-acting nucleotide sequences
of two regulatory elements
1) Promoters - bind TFs and RNA
Pol - Promoters are necessary
for transcription initiation.
2) Enhancers - only bind TFs. TFs
bend or loop the DNA. This brings
Distant Enhancers and Promoters
into close proximity to form
activated transcription complexes increasing the overall transcriptional
rate. See Fig. 1.
RNA
Pol II
TF
TF
5'
Fig. 1
3'
RNA
Pol II
TF
TF
5'
In the Promoter region, the modular elements, TATA box, CAAT box, and GC box,
bind TFs in order to stimulate transcription. See Fig. 2
Fig. 2
Pre-mRNA being synthesized by RNA Pol II
Promoter (30 – 120 bp long) – upstream of transcriptional start site
GC
box
CAAT
box
TATA
box
3' GGGCGG
GGCCAATC
TATAAA
-110
bp
-70
bp
Transcription
start site
Pre-mRNA 5'
gene
-30
bp
+1
bp
Enhancers greatly increase the efficiency of transcription initiation. In contrast to
Promoters, Enhancers have the following properties: i) Their positions and orientations
need not be fixed, ii) They are at a greater distance from the target gene than are
Promoters, and iii) They generically enhance the transcription of surrounding genes.
Termination:
1) rRNA (RNA Pol I) - Modified rho-independent. Strange G-C
dyad region (many A's and U's
intervening), followed by 26 A's,
followed by 16 bases of any kind,
followed by UCCACCUGGUC, followed
by 3 bases, followed by AGGC.
2) pre-mRNA (RNA Pol II) - termination mechanism unknown.
Processing begins before transcription
ends. Possibly, processing proteins
may literally pull pre-mRNA away and
force RNA Pol II off of DNA.
3) tRNA (RNA Pol III) - standard rho-independent.
Three major aspects
of post-transcriptional processing
of primary transcripts of pre-mRNAs in eukaryotes.
RNA splicing - removal of non-coding intron (intervening)
sequences, between coding exon (expressed) sequences.
Capping of 5' end (i.e., 7-methylquanosine) and addition of poly A
tail to 3' end of pre-mRNA.
In eukaryotes, pre-mRNA must associate with proteins to get out of
the nucleus and into the cytoplasm.
Four major aspects of post-transcriptional
processing of tRNAs in eukaryotes.
Cut at G on 5' end.
Addition of CCA sequence at 3' end (where AA will attach).
Base modifications (i.e., thymine, pseudouracil) in looped regions.
Introns excised from terminal loop.
Aspects of post-transcriptional
processing of rRNAs in eukaryotes.
Introns removed by autocatalytic activity of EXON regions.
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Exon 1
Intron A
Exon 2
Intron B
Exon 3
Pulse-Chase tracking of transcription in Eukaryotes:
Transport of RNA to the cytoplasm from the nucleus of
eukaryotic cells can be determined through pulse-chase studies
using 3H-uridine. If a eukaryotic cell is exposed to radioactive
RNA precursor for a few minutes, and the intracellular location
of the incorporated radioactivity is determined by
autoradiography, almost all of the nascent, labeled RNA is
found in the nucleus. If the short exposure “pulse” to the
labeled RNA is followed by a period of growth in non-radioactive
medium before autoradiography, almost all of the radioactivity
is found in the cytoplasm.