3' RNA Structure and Transcription

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3. RNA Structure and Transcription
a). Chemistry of RNA i). Bases found in
RNA ii). Ribose sugar iii). RNA
polynucleotide chain iv). Secondary and
tertiary structure b). Characteristics of
prokaryotic RNA i). Classes of prokaryotic
RNA ii). Structure of prokaryotic messenger
RNA c). Transcription initiation in
prokaryotes i). Transcription ii). Promoter
structure iii). Prokaryotic RNA polymerase
structure iv). Initiation of transcription and
the sigma cycle d). Regulation of the lactose
operon i). Function of the lactose
operon ii). Negative control Lac repressor and
inducer iii). Positive control CAP and cAMP
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The major bases found in DNA and RNA
DNA RNA Adenine Adenine
Cytosine Cytosine Guanine Guanine
Thymine Uracil (U)
uracil-adenine base pair
thymine-adenine base pair
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Examples of modified bases found in RNA
Dihydrouridine
Pseudouridine
1-methylguanosine
7-methylguanosine
1-methyladenosine
2-thiocytidine
5-methylcytidine
Ribothymine
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RNA polynucleotide chain

• 2 -OH makes
• 3, 5 phosphodiester
• bond unstable

DNA polynucleotide chain
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Secondary structure
Tertiary structure
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Classes of prokaryotic RNA

• ribosomal RNA (rRNA)
• 16S (small ribosomal subunit)
• 23S (large ribosomal subunit)
• 5S (large ribosomal subunit)
• transfer RNA (tRNA)
• messenger RNA (mRNA)

Structure of prokaryotic messenger RNA
Shine-Dalgarno sequence
initiation
PuPuPuPuPuPuPuPu
AUG
5
translated region
AAU
3
termination
The Shine-Dalgarno (SD) sequence base-pairs with
a pyrimidine-rich sequence in 16S rRNA to
facilitate the initiation of protein synthesis
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Transcription
closed promoter complex
RNA polymerase
open promoter complex
initiation
elongation
termination
RNA product
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Promoter structure in prokaryotes
mRNA
PuPuPuPuPuPuPuPu
AUG
5
-30
-10
1


Promoter
transcription start site
mRNA
5
-30 region
-10 region
TTGACA
TATAAT
AACTGT
ATATTA
1
20
-7
-12
-31
-36
Pribnow box
consensus sequences
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Prokaryotic RNA polymerase structure
RNA polymerase of bacteria is a multisubunit
protein Subunit Number Role a
2 uncertain b (Rifampicin target) 1 forms
phosphodiester bonds b 1 binds DNA
template s 1 recognizes promoter
and facilitates initiation
a2bbs
a2bb s
holoenzyme core polymerase
sigma factor
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• The function of sigma factor
• the sigma subunit of RNA polymerase is an
  initiation factor
• there are several different sigma factors in E.
  coli that are
• specific for different sets of genes
• sigma factor functions to ensure that RNA
  polymerase binds
• stably to DNA only at promoters
• sigma destablizes nonspecific binding to
  non-promoter DNA
• sigma stabilizes specific binding to promoter
  DNA
• this accelerates the search for promoter DNA

• Ka (M-1)
• Any DNA Promoter DNA
• (nonspecific) (specific)
• Core 2 X 1011
• Holo 1 X 107 1013 to 1015
• promoters vary in strength by two orders of
  magnitude

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• closed promoter complex (moderately stable)
• the sigma subunit binds to the -10 region

s
RNA polymerase holoenzyme ( s factor)

• open promoter complex (highly stable)
• the holoenzyme has very high affinity for
• promoter regions because of sigma factor

s

• once initiation takes place, RNA polymerase does
• not need very high affinity for the promoter
• sigma factor dissociates from the core
  polymerase
• after a few elongation reactions

s

• elongation takes place with
• the core RNA polymerase

• sigma can re-bind
• other core enzymes

The sigma cycle
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Mechanism of RNA synthesis
RNA
RNA
A T
A T
U A
U A

• RNA synthesis usually initiated with ATP or GTP
  (the first nucleotide)
• RNA chains are synthesized in a 5 to 3
  direction

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The lactose operon in E. coli

• promoter binds CAP and RNA polymerase
• operator binds the lac repressor

promoter - operator
lac I
P
O
lac Z
lac Y
lac A
lac repressor
b-galactosidase
permease
acetylase

• the function of the lactose (lac) operon is to
  produce the enzymes
• required to metabolize lactose for energy when
  it is required by the cell

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Regulation of the lactose operon - negative
control
promoter - operator
lac I
P
O
lac Z
lac Y
lac A
lac repressor

• the repressor tetramer binds to the operator and
  prevents
• RNA polymerase from binding to the promoter

lac I
P
lac Z
lac Y
lac A
NO TRANSCRIPTION
RNA pol

• RNA polymerase is blocked from the promoter

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Alleviation of negative control - action of the
inducer of the lac operon

• when lactose becomes available, it is taken up
  by the cell
• allolactose (an intermediate in the hydrolysis
  of lactose) is produced
• one molecule of allolactose binds to each of the
  repressor subunits
• binding of allolactose results in a
  conformational change in the repressor
• the conformational change results in decreased
  affinity of the repressor
• for the operator and dissociation of the
  repressor from the DNA

allolactose
lac I
P
lac Z
lac Y
lac A
lac I
P
lac Z
lac Y
lac A
NO TRANSCRIPTION

• IPTG (isopropyl thiogalactoside)
• is also used as a (non-physiological) inducer

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• repressor (with bound allolactose) dissociates
  from the operator
• negative control (repression) is alleviated,
  however...

lac I
P
lac Z
lac Y
lac A
O
NO TRANSCRIPTION
RNA pol

• RNA polymerase cannot form a stable complex with
  the promoter

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Affinity of lac repressor for DNA (M-1)
DNA Repressor Repressor
Inducer lac operator 2 X 1013 2 X 1010 All
other DNA 2 X 106 2 X 106 ____________________
_______________________________ Specificity1
107 104 ____________________________________
_______________ 1 Specificity is the ratio of
(Ka for binding to operator DNA) / (Ka for
binding to random DNA)
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Regulation of the lactose operon - positive
control

• in the presence of both lactose and glucose it
  is not necessary
• for the cell to metabolize lactose for
  energy
• in the absence of glucose and in the presence of
  lactose it becomes
• advantageous to make use of the available
  lactose for energy
• in the absence of glucose cells synthesize
  cyclic AMP (cAMP)
• cAMP1 serves as a positive regulator of
  catabolite operons (lac operon)
• cAMP binds the dimeric cAMP binding protein
  (CAP)2
• binding of cAMP increases the affinity of CAP
  for the promoter
• binding of CAP to the promoter facilitates the
  binding of RNA polymerase
• 1 cAMP 3, 5 cyclic adenosine monophosphate

active CAP
inactive CAP
cAMP

lac I
P
O
lac Z
lac Y
lac A
NO TRANSCRIPTION
2 also termed catabolite activator protein
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Activation of lac operon transcription
lac I
lac Z
lac Y
lac A
lac repressor
TRANSCRIPTION AND TRANSLATION OCCUR
b-galactosidase
permease
acetylase
inactive repressor

• the function of the lactose (lac) operon is to
  produce the enzymes
• required to metabolize lactose for energy when
  it is required by the cell