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(GENE EXPRESSION)

TRANSLATION, AND REGULATION


Sismindari, Ph.D.Prof. Kuliah ke 6 Farmasi UGM
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Heredity -the transmission of characters to progeny. DNA carries the information necessary for the transmission of characters. The biological information is encoded in the sequence of bases. TB
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I. RNA (ribonucleic acid) A polymer of nucleosides held together by phosphodiester bonds. RNA is usually single-stranded. RNA plays a key role in decoding the information in DNA.
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A. Functions of the major RNAs


1. messenger RNAs (mRNA) contain genetic information to encode a protein
phe

2. transfer RNAs (tRNA) act as adapters between the mRNA nucleotide code and amino acids during protein synthesis

3. ribosomal RNAs (rRNA) are structural and catalytic component of ribosomes


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4. Complementary base pairing CCCTTTGGGAAA


hydrogen bonding

DNA

GGGAAACCCUUU
GGGAAACCCUUU

RNA
RNA

CCCUUUGGGAAA

RNA
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5. RNA stem loops


A common RNA secondary structure

ssRNA

complementary base pairing (helical)


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II. TRANSCRIPTION Most commonly, gene expression refers to the decoding of genes into proteins or RNAs.

1 gene encodes 1 polypeptide, 1 tRNA, 1 rRNA, or 1other RNA

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A. Gene numbers

group viruses prokaryotes eukaryotes

approximate gene number

4-200 500-12,000 5,000-125,000


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Any given species has a unique set of genes that confers a unique set of properties. Proteins and RNAs determine all of the characteristics of organisms and cells.
Example: Escherichia coli has 4405 genes
~117 encode RNAs (tRNA, rRNA) ~4288 encode proteins TB
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C. Gene expression in prokaryotes 1. Expression of single genes


Ex.1: a single gene that encodes a protein
1 gene

transcription translation
1 mRNA

1 polypeptide

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Ex. 2: a single gene that encodes one rRNA or tRNA

1 gene
transcription

1 RNA
RNA processing

degraded

1 tRNA etc.
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2. Expression of operons
operon two or more genes transcribed together
A B C DNA

transcription

polycistronic message polycistronic mRNA a single RNA molecule that represents more than one gene
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a. Operons can encode several polypeptides or proteins.


A B C

1 operon

transcription

1 polycistronic mRNA
translation
B

2 or more polypeptides

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b. Operons can encode several rRNA molecules.


1 operon 1 polycistronic RNA processing rRNA rRNA degraded

2 or more rRNAs TB
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3. Important points

The details of organization, processing and degradation are different for different RNAs. Most prokaryotes use operons. Operons are used to coordinate gene expression and often contain genes of related function.

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D. Eukaryotic gene expression 1. Expression of eukaryotic rRNA and tRNA genes

The expression of rRNA and tRNA is similar in eukaryotes and prokaryotes.


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2. Eukaryotic protein expression


a. Typical eukaryotic genes have exons and introns.

E I

E
gene

E = exon = coding sequences I = intron = intervening, noncoding sequences

Eukaryotes do NOT have operons

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1 gene with exons and introns


E I E I E I E

transcription

1 RNA representing exons and introns (primary transcript)

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b. Primary transcripts primary transcript processing 1 mRNA

1 polypeptide

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c. Processing of primary transcripts i. capping ii. splicing iii. tailing

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i. Capping Addition of a 5' cap

CAP Capping usually occurs before transcription is finished.

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Typical 5' CAP


P

CH3

O N N

OCH2 N O

NH2

HO OH

5' carbon of RNA chain

7-methylguanosine 5' to 5' linkage


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Know the name (methylguanosine cap, 5' cap), but don't memorize structure.

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ii. Splicing The removal of introns.

primary transcript splicing


RNA without introns

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iii. Tailing

Addition of a poly-A tail

A1A2...A~200
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3. Notes on eukaryotic RNA processing

The exact order of capping, tailing and splicing varies for different genes. Processing occurs in the nucleus Poly-A tails are added by poly-A polymerase, NOT during transcription.
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TRANSLATION

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III Prokaryotic translation 1. Key components of translation 2. Steps in translation 3. The genetic code

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Overview of prokaryotic translation


Protein synthesis from an mRNA template.
translated region mRNA
phe

translation

protein of specific amino acid sequence


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1. Key components of translation


A. mRNA B. tRNA C. ribosomes and rRNA

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A. mRNA RNA template for protein synthesis


translated region Shineseries of codons Dalgarno (usually ~300 codons) sequence
mRNA start codon stop codon
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1. Shine-Dalgarno sequence
~AGGAGG, ribosome binding sequence, critical for ribosome binding

2. start codons AUG, GUG, or UUG


3. stop codons (nonsense codons) UAA, UGA, or UAG
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4. Translated region (coding sequence) Series of codons that determines the amino acid sequence of the encoded protein.
Coding sequences have an average of about 300 codons. Except for the stop codon, each codon specifies a particular amino acid.

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5. Codons consist of 3 bases


start codon

codons

2 3 4 1 AUGCAUUGUUCU...
fMet - His - Cys - Ser ... 1 2 3 4 TB
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protein

B. tRNA The adapter molecule for translation

1. Particular tRNAs carry particular amino acids.


f-Met
His

His

tRNA-f-Met

tRNA-His

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2. Particular tRNAs recognize particular codons. codons AUGCAUUGUUCU...


tRNAs

AA1

AA2

amino acid (AA)


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This allows amino acids to be brought TB together in a particular order.

3. tRNA structure All tRNAs are generally similar in structure. a. 1o structure ssRNA 73-93 nucleotides long 5'
UAC

3'

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b. structure clover leaf

o 2

acceptor arm D-arm


extra arm anticodon loop
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TYC arm

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c. 3o structure inverted "L"

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d. Anticodon
A 3 base sequence in tRNA complementary to a specific codon.

anticodon
Base pairing between an anticodon and a codon allows a tRNA to recognize a specific codon. TB
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e. codon-anticodon interactions
anticodon

3'
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5'
tRNA

5'

UUA AAU codon


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3' mRNA
TB

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4. tRNA charging (adding amino acid)


3' O H2N-CH-C-O 3' R

tRNA (uncharged)

aminoacyl-tRNA (charged)
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tRNA charging uses the energy of ATP TB

Aminoacyl-tRNA synthetases

enzymes that attach amino acids to tRNA

enzyme ATP amino acid

aminoacyl-AMP

tRNA
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aminoacyl-tRNA AMP PPiPPi = inorganic pyrophosphate TB AMP = adenosine monophosphate

5. tRNA facts

Prokaryotes have about 60 different tRNAs.


tRNAs contain many modified bases.

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C. Ribosomes and rRNA Ribosomes ribonucleoprotein complexes that catalyze protein synthesis. rRNAs have structural and catalytic roles
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1. Prokaryotic 70s ribosome 23s rRNA 5s rRNA 34 proteins 16s RNA 21 proteins

50s subunit
30s subunit
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2. Ribosomal sites where tRNAs bind E = exit

E
P A

P = peptidyl A = aminoacyl
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3. 16S rRNA
The 3' end of the 16s rRNA is complementary to the ShineDalgarno sequence (ribosome binding sequence of mRNAs)

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II. Steps in translation A. initiation


P-site
f-met
AGGAGG-----AUG

30s subunit of ribosome


mRNA

Shine-Dalgarno
(AGGAGG on mRNA) f-met

AUG GTP hydrolysis 50s subunit 30s subunit TB


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1. f-met tRNA (formyl-methionine tRNA)


In Bacteria, different met-tRNAs are used for elongation and initiation.
met tRNA

initiation, formyl-methionine elongation, methionine


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met tRNA

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2. Initiation in different domains


In Bacteria, the formyl group of the initiator formylmethionine (f-met) is later removed. In Eukarya and Archaea, initiation begins with methionine rather than f-met.
In Eukarya, the ribosome recognizes the 7-methylguanosine cap at the 5 end of mRNA and initiates at the first AUG.
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B. Elongation
1. AA-tRNA binding
AA mRNA P-site A-site

AA

AA AA
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2. peptide bond synthesis


AA AA

(peptidyl transferase)
AA AA

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3. translocation
AA
AA GTP hydrolysis

AA

AA

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C. Termination
AA AA AA AA stop codon

AA
UAA

termination
AA AA AA AA

AA
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D. Additional notes on translation 1. Ribosomes move along the mRNA. mRNAs can be translated by 5-10 ribosomes simultaneously. mRNA

"Polysomes" are mRNAs with several ribosomes attached.

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2. In prokaryotes only, transcription and translation are coupled. Translation begins before transcription ends. DNA
mRNA
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3. Protein folding into the active form can occur spontaneously or with the help of a large protein complex called a molecular chaperone.
ATP ADP
improperly folded protein molecular chaperone

properly folded protein

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III. The genetic code


A. universal code B. degenerate code 1. synonyms 2. codon families 3. codon pairs C. wobble base pairing
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III. The genetic code


8 codon families, 14 codon pairs, 3 stop codons

(Do not 493 memorize)

A. The genetic code is almost universal. Most organisms use the same genetic code.

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B. The genetic code is degenerate. more than one codon can code for the same amino acid

UUU phenylalanine UUC phenylalanine


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1. synonyms codons that code for the same amino acid UUU phenylalanine UUC phenylalanine Not all synonyms are used with equal frequency. This is called "codon usage bias".

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2. codon families any nucleotide in the 3rd positions leucine

CUU CUC CUA CUG

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3. codon pairs UUU UUC CAA CAG

any pyrimidine in the 3rd position

phenylalanine
glutamine
any purine in the 3rd position

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C. Wobble base pairing


U-G and G-U base pairs are allowed in the 3rd position of the codon.

codon (mRNA) 5' 3'

UUU AAG
anticodon (tRNA)

3' 5'
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Regulation of Gene Expression

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Regulation of Gene Expression I: I. Regulation of gene expression II. Transcriptional regulation III. Examples of gene repression IV. Example of gene induction

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I. Regulation of gene expression Not all genes are turned on (expressed) all the time In general, they are turned on only when needed.
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Cells can respond to environmental changes by regulating gene expression.


maltose arginine

lactose

glucose

tryptophan
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Different genes are expressed when cells grow on different compounds.


glucose
e.g. Growth on lactose requires expression of at least three additional genes.

maltose

TCA

lactose
(galactose--1,4-glucose)

P O

lacZ
-galactosidase

lacY

lacA
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lac permease (transport protein)

A. Why regulate gene expression? Regulation allows cells to respond to environmental conditions by synthesizing selected gene products only when they are needed.

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B. Gene expression synthesis of a gene product


1. constitutive 2. regulated

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1. Constitutive gene expression expression of genes at about the same level under all environmental conditions
e.g. "housekeeping genes" like primase ssDNA binding proteins
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2. Regulated gene expression Control of the rate of protein or RNA synthesis as an adaptive response to stimuli.
induction: increase in gene expression

repression: decrease in gene expression


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a. gene induction increase in gene expression


inducer

amount of gene product

time
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e.g. genes that encode enzymes for tryptophan biosynthesis are repressed by tryptophan.
tryptophan absent
enzymes for tryptophan biosynthesis (molecules/cell)

tryptophan present

time

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Important general principle catabolic substrates (e.g. maltose and lactose) induce the genes required for their catabolism biosynthetic molecules (e.g. amino acids and purines) repress the genes required for the biosynthesis
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II. Transcriptional regulation

regulation of RNA synthesis the most common method of gene regulation in all cells
A. Regulatory proteins B. Regulatory protein binding sites C. Effector molecules
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A. Regulatory proteins
Transcriptional regulation is mediated by regulatory proteins. Cells have many different regulatory proteins. Specific regulatory proteins control the transcription of specific groups of genes. Examples of regulatory proteins are "repressor proteins" and "activator proteins."
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1. Repressor proteins
Repressor protein (dimer)

DNA
RNA polymerase Promoter Repressor proteins decrease transcription when bound to DNA by interfering with the TB activity or binding of RNA polymerase.
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2. Activator proteins
Activator protein

DNA

RNA polymerase "weak" promoter Activator proteins increase transcription when bound to DNA by helping RNA polymerase bind TB to weak promoters.
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B. Regulatory protein binding sites Regulatory proteins bind to specific DNA sequences. A particular regulatory protein will only control the expression of genes having appropriate binding sites.
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1. Operator sites
binding sites for repressor proteins GTGTAAACGATTCCAC
lac repressor binding site

CACATTTGCTAAGGTG
Imperfect palindrome

Usually found near promoters.

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2. Activator binding sites Binding sites for activator proteins

GTGAGTTAGCTCAC CACTCAATCGAGTG Imperfect palindrome

crp binding site

Usually found near promoters.

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C. Effector molecules Small molecules from the environment (or made inside cells) that signal specific changes in gene expression.

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1. Classes of effectors
a. inducers
maltose

small molecules that mediate gene induction


e.g. catabolic substrates: sugars, amino acids, fatty acids
lactose
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b. corepressors small molecules that mediate gene repression e.g. biosynthetic products: amino acids, purines, pyrimidines, fatty acids etc.
arginine tryptophan
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2. How effectors work


Effectors change the DNA binding affinity of regulatory proteins for their binding sites.
regulatory protein

effector

conformational change (change in 3-D structure)


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A. Some effectors increase DNA binding affinity


regulatory protein conformational change (change in 3-D structure)

DNA
effector
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B. Some effectors decrease DNA binding affinity DNA

regulatory protein

conformational change (change in 3-D structure)

effector

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Since most regulatory proteins influence transcription when bound to DNA, the binding of effectors to regulatory proteins changes gene expression.
regulatory protein effector
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III. Examples of gene repression A. Regulation of the trp operon B. Regulation of the arg operon

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A. The trp operon is a group of genes used for biosynthesis of the amino acid tryptophan (Trp). The trp operon promoter trp genes

polycistronic mRNA

Five enzymes for tryptophan biosynthesis TB


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1. When Trp is NOT available in the environment, expression of the trp operon allows Escherchia coli to make Trp needed for protein synthesis.
2. When Trp is available, E. coli takes up Trp from the environment and represses the trp operon.

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trp promoter inactive repressor

operator

tryptophan active repressor

RNA polymerase

genes on

genes off

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Note: Repression of the trp operon by tryptophan involves a repressor protein.


When tryptophan binds to the repressor protein, the repressor protein binds to DNA. Transcription is blocked.

Result: VERY low amounts of tryptophan are synthesized when the cell can get tryptophan from the environment .
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B. Regulation of the arg operon for arginine biosynthesis


If arginine is present in large amounts arg biosynthetic enzymes NOT needed arg binds repressor arg-repressor binds DNA RNA polymerase can't bind to promoter
argC argB argH operator arg biosynthetic genes transcription rate decreases P

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If arg is absent, the cell needs to make arg repressor doesn't bind DNA RNA polymerase can bind transcription of arg genes occurs
P operator argC argB argH arg biosynthetic genes

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IV. Example of gene induction: Regulation of the lac operon


A. The lac operon is a group of genes used for catabolism of the sugar lactose.
promoter
Z operator
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lac genes
Y A
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When lactose is unavailable, the catabolic enzymes are NOT needed. The lac operon is expressed at only very low levels.

When lactose is available, E. coli induces expression of lac operon.

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B. Lactose unavailable
lac promoter Z Y A

genes off

In the ABSENCE of lactose, the lac repressor protein binds DNA.


Note: the role of crp/cAMP in control of the lac operon is not considered here. TB
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C. Lactose available
lac promoter

Z
RNA polymerase lactose allolactose

genes on

repressor does not bind DNA TB


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Important points

Repressor proteins can mediate gene repression (e.g. trp operon) or gene induction (lac operon).
Activator proteins can mediate both gene induction and gene repression.
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Some repressor proteins mediate gene induction. Example: the lac repressor
Lactose (a sugar) can be an energy source. If lactose is absent, enzymes for using lactose are not needed lac repressor binds to the lac operator the lac genes are not expressed
CAP site P O lacZ lacY lacA
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Some repressor proteins mediate gene induction.


Lactose ( ) induces the expression of lac genes. If lactose is present, + enzymes for using lactose are needed (allo)lactose binds to the lac repressor and causes a conformational change repressor-lac does NOT bind to DNA expression of lac genes is possible
CAP site P O lacZ lacY lacA
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D. The catabolite activator protein


1. In the lac operon, the activator protein is called the catabolite activator protein (CAP) or the cyclic AMP receptor protein (crp). 2. When cyclic AMP (cAMP) is present, the cAMP/CAP (crp) complex binds DNA and activates transcription.
CAP (crp) cAMP

cAMP/CAP complex binds DNA TB


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NH2 N N N
(Don't memorize)

O
HOP=O

CH2

OH
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cyclic AMP (cAMP) cyclic adenosine monophosphate

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Role of CAP (crp) in the lac operon


Without activator protein, RNA polymerase binds weakly and the transcription rate is low.
crp P O binding site lacZ lacY lacA

With activator protein (crp), RNA polymerase binds well and the transcription rate is higher.
P O lacZ lacY lacA
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3. MANY operons that encode catabolic enzymes have the same crp binding site ( ) and are controlled by the same regulatory protein (CAP or crp).
crp binding site

bacterial chromosome

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1. Catabolite repression enables Escherichia coli to use glucose in preference to other glucose carbon sources.
Lactose utilization requires additional proteins. crp P O binding site lacZ lacY

maltose

TCA

lactose
(galactose--1,4-glucose)

lacA
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-galactosidase

lac permease (transport protein)

2. Key components of catabolite repression a. cAMP (cyclic AMP)

an effector molecule that increases the DNA binding affinity of the catabolite activator protein Catabolite activator protein, a transcriptional regulatory protein; also called crp (cAMP receptor protein) TB
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b. CAP (or crp)

3. CAP/cAMP binds to DNA and regulates transcription.

CAP (or crp)


cAMP
cAMP/CAP complex

CAP (or crp) binding sites

bacterial chromosome

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4. How does catabolite repression work?


a. Genes needed for the catabolism of many carbon and energy sources require cAMP/CAP for expression.

*b. Glucose decreases cellular cAMP levels.


c. Without cAMP/CAP, genes required to catabolize nonglucose energy sources are transcribed at very low rates. d. Therefore, glucose is preferentially used as a carbon and energy source.

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C. Global regulation is often used together with other more specific regulatory systems. Example: the lactose operon requires both lactose and cAMP/CAP for induction.
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Both lactose and cAMP/CAP are needed for high induction of lac operon.
glucose decreases cAMP glucose present, lactose absent

crp P O lacZ lacY lacA binding site lac repressor binds DNA in absence of lactose

P O

lacZ

lacY

lacA
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glucose absent, lactose present

III. Two-component regulatory systems


Transcriptional regulatory systems composed of a sensor kinase and response regulator.

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A. Sensor kinase Integral membrane proteins that sense environmental conditions and phosphorylate proteins

B. Response regulator
Cytoplasmic transcriptional regulatory proteins controlled by sensor kinases through phosphorylation TB
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sensor kinase

effector

P
phosphorylation

dephosphorylation

P P

response regulator

cytoplasmic membrane
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