RNA Structure and Transcription

8/9/2019 RNA Structure and Transcription

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RNA Structure a

a). Chemistry of RNA

i). Bases found in Rii). Ribose sugar

iii). RNA polynucleo

iv). Secondary and t

b). Characteristics of prok

i). Classes of prokaii). Structure of prok

c). Transcription initiation i

i). Transcription

ii). Promoter structu

iii). Prokaryotic RNiv). Initiation of tran

d). Regulation of the lacto

i). Function of the la

ii). Negative control:

iii). Positive control:

d

Transcription

A

ide chain

ertiary structure

ryotic RNA

yotic RNAaryotic messenger RNA

n prokaryotes

re

polymerase structurecription and the sigma cycle

e operon

ctose operon

Lac repressor and inducer

CAP and cAMP



The major bases fou

DNA

Adenine

Cytosine

Guanine

Thymine

thymine-adenine base pair

d in DNA and RNA

RNA

Adenine

Cytosine

Guanine

Uracil (U)

uracil-adenine base pair



Examples of modified

Dihydrouridine Pseudouridine 1-

1-methyladenosine 2-thiocytidine

b

ases found in RNA

ethylguanosine 7-methylguanosine

5-methylcytidine Ribothymine



DNA polynucleotide chain

RNA polynucleotide chain

2· -OH makes

3·, 5· phosphodiester

bond unstable



Secondary structure

Tertiary structure





RNA p

closed promoter

open promoter

initiation

R

Transcription

lymerase

complex

omplex

elongation

termination

A product



Promoter structure in pro

5¶

Promoter

-31-36

TTGACA

AACTGT

-30 region

84 79 53 45%82

T T G64

A C A 79

T

consensus seque

-30 -10

transcriptio

+1

[

aryotes

PuPuPuPuPuPuPuPu AUG

+1 +20-7-12

5¶mRNA

mRNA

TATAAT

ATATTA

-10 region

44

T96%

T95

A 59

A 51

A

ces

start site

Pribnow box

]



Prokaryotic RNA polymera

RNA polymerase of bacteria i

Subunit Number

E 2

F(Rifampicin target) 1 F¶ 1

W 1

E FF¶W

holoenzyme core p

e structure

a multisubunit protein

Role

uncertain

forms phosphodiester bondsbinds DNA template

recognizes promoter and

facilitates initiation

FF¶ + W

olymerase sigma factor



The function

the sigma subunit of RNA polyme

there are several different sigma f

specific for different sets of

sigma factor functions to ensu

stably to DNA only at pro

sigma destablizes nonspecific

sigma stabilizes specific bindi

this accelerates the search for

K

Any DNA

(nonspecific)

Core 2 X 1011

Holo 1 X 107

promoters vary in ³strength´ b

f sigma factor

rase is an ³initiation factor´

actors in E. coli that are

enes

e that RNA polymerase binds

oters

binding to non-promoter DNA

g to promoter DNA

promoter DNA

(M-1)

Promoter DNA

(specific)

1013 to 1015

~two orders of magnitude



RNA pol

closed promote

the sigma subu

once initiation t

not need very h

sigma factor di

after a few elon

open promoter

the holoenzyme

promoter regio

W

sigma can re-bind

other core enzymes

W

Wmerase holoenzyme (+

Wfactor)

r complex (moderately stable)

it binds to the -10 region

kes place, RNA polymerase does

igh affinity for the promoter

sociates from the core polymerase

gation reactions

elongation takes place with

the core RNA polymerase

omplex (highly stable)

has very high affinity for

s because of sigma factor

The sigma cycle



Mechanism of

RNA synthesis usually initiated wi

RNA chains are synthesized in a 5

A = T

U = A

RNA

NA synthesis

h ATP or GTP (the first nucleotide)

¶ to 3¶ direction

A = T

U = A

RNA



lac I P O

promoter - operator

lac repressor

la

The lactose operon in E. col

F-galact

LACTOSE

F-galactosidase

the function of the lactose (lac) o

required to metabolize lactose for e

promoter binds CA

operator binds the l

Z lac Y lac A

i

sidase permease acetylase

GLUCOSE + GALACTOSE

eron is to produce the enzymes

ergy when it is required by the cell

and RNA polymerase

ac repressor



Regulation of the lactose operon - neg

lac I P O

promoter - operator

lac repressor

lac I P la

the repressor tetra

RNA polymerase

la

RNA pol RNA polymerase is

tive control

Z lac Y lac A

er binds to the operator and prevents

from binding to the promoter

Z lac Y lac A

NO TRANSCRIPTION

blocked from the promoter



when lactose becomes available, it i

allolactose (an intermediate in the h

one molecule of allolactose binds to

binding of allolactose results in a co

the conformational change results in

for the operator and dissociation of

Alleviation of negative control - action

allola

lac I P la

lac I P la

IPTG (isopropyl thiogalactoside)

is also used as a (non-physiological)

NO TRANSCRIPTION

taken up by the cell

drolysis of lactose) is produced

each of the repressor subunits

formational change in the repressor

decreased affinity of the repressor

he repressor from the DNA

f the inducer of the lac operon

tose

Z lac Y lac A

Z lac Y lac A

inducer



lac I P la

RNA pol

O

repressor (with bound allolactose)

negative control (repression) is all

RNA polymerase cannot form a sta

Z lac Y lac A

NO TRANSCRIPTION

dissociates from the operator

viated, however...

ble complex with the promoter



Affinity of lac repr

DNA Repressor

lac operator 2 X 1013

All other DNA 2 X 106

_________________________

Specificity1 107

_________________________

1 Specificity is the ratio of

(Ka for binding to operator DNA

ssor for DNA (M-1)

Repressor + Inducer

2 X 1010

2 X 106

_________________________

104

_________________________

) / (Ka for binding to random DNA)



lac I P O la

Regulation of the lactose operon - posi

in the presence of both lactose and g

for the cell to metabolize lactose fo

in the absence of glucose and in theadvantageous to make use of the a

in the absence of glucose cells synth

cAMP1 serves as a positive regulator

cAMP binds the dimeric cAMP bindin

binding of cAMP increases the affinit

binding of CAP to the promoter facilit

1 cAMP = 3¶, 5¶ cyclic adenosine monophospha

active CAP

2 also termed catabolite activator protein

Z lac Y lac A

ive control

lucose it is not necessary

energy

resence of lactose it becomesailable lactose for energy

esize cyclic AMP (cAMP)

of catabolite operons (lac operon)

g protein (CAP)2

of CAP for the promoter

ates the binding of RNA polymerase

te

inactive CAPcAMP

+

NO TRANSCRIPTION



lac I

lac repressor

la

F-galact

RNA pol

TRANSC

inactive repressor

Activation of lac operon transcription

the function of the lactose (lac)

required to metabolize lactose for e

Z lac Y lac A

sidase permease acetylase

RIPTION AND TRANSLATION OCCUR

peron is to produce the enzymes

nergy when it is required by the cell

Documents

RNA Structure and Transcription