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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
The major bases found in DNA and RNA

DNA RNA

Adenine Adenine
Cytosine Cytosine
Guanine Guanine
Thymine Uracil (U)

thymine-adenine base pair uracil-adenine base pair


Examples of modified bases found in RNA

Dihydrouridine Pseudouridine 1-methylguanosine 7-methylguanosine

1-methyladenosine 2-thiocytidine 5-methylcytidine Ribothymine


RNA polynucleotide chain
• 2’ -OH makes
3’, 5’ phosphodiester
bond unstable

DNA polynucleotide chain


Secondary structure

Tertiary structure
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
5’ PuPuPuPuPuPuPuPu AUG
translated region
3’ AAU
termination

The Shine-Dalgarno (SD) sequence base-pairs with a pyrimidine-rich


sequence in 16S rRNA to facilitate the initiation of protein synthesis
Transcription
closed promoter complex

RNA polymerase
open promoter complex

initiation

elongation

termination

RNA product
Promoter structure in prokaryotes

mRNA
5’ PuPuPuPuPuPuPuPu AUG
-30 -10 +1
[ Promoter
]
transcription start site
mRNA
5’
-30 region -10 region
TTGACA TATAAT
AACTGT ATATTA
-36 -31 -12 -7 +1 +20
Pribnow box
T T G AC A TATA AT
82 84 79 64 53 45% 79 95 44 59 51 96%

consensus sequences
Prokaryotic RNA polymerase structure

RNA polymerase of bacteria is a multisubunit protein

Subunit Number Role

 2 uncertain
(Rifampicin target) 1 forms phosphodiester bonds
’ 1 binds DNA template
 1 recognizes promoter and
facilitates initiation

’ ’ + 
holoenzyme core polymerase sigma factor
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


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

RNA polymerase holoenzyme (+  factor)

• open promoter complex (highly stable)


• the holoenzyme has very high affinity for
promoter regions because of sigma factor

• 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

 • elongation takes place with


the core RNA polymerase

• sigma can re-bind


other core enzymes The sigma cycle
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
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 -galactosidase permease acetylase

-galactosidase
LACTOSE GLUCOSE + GALACTOSE

•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
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


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

lac I P O lac Z lac Y lac A

NO TRANSCRIPTION

RNA pol

• RNA polymerase cannot form a stable complex with the promoter


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


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1
Specificity is the ratio of
(Ka for binding to operator DNA) / (Ka for binding to random DNA)
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 cAMP inactive CAP

lac I P O lac Z lac Y lac A

2
also termed catabolite activator protein NO TRANSCRIPTION
Activation of lac operon transcription

RNA pol
lac I lac Z lac Y lac A

lac repressor TRANSCRIPTION AND TRANSLATION OCCUR

-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

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