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TRANSLATION, AND REGULATION

(GENE EXPRESSION)

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.

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I. RNA (ribonucleic acid)

A polymer of nucleosides held together by phosphodiester bonds.

RNA plays a key role in decodingthe information in DNA.

RNA is usually single-stranded.

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A. Functions of the major RNAs

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

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

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

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4. Complementary base pairing

CCCUUUGGGAAA

GGGAAACCCUUU RNA

RNA

GGGAAACCCUUU RNA

CCCTTTGGGAAA DNA

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hydrogenbonding

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5. RNA stem loops

complementarybase pairing(helical)ssRNA

A common RNA secondary structure

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II. TRANSCRIPTIONMost 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 TB

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A. Gene numbers

virusesprokaryotes eukaryotes

groupapproximategene number

4-200500-12,000 5,000-125,000

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Any given species has a unique setof genes that confers a unique set of properties.

Proteins and RNAs determine all of thecharacteristics of organisms and cells.

Example: Escherichia coli has 4405 genes

~117 encode RNAs (tRNA, rRNA) ~4288 encode proteins TB

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1 gene

1 mRNA

transcription

1 polypeptide

translation

1. Expression of single genesEx.1: a single gene that encodes a protein

C. Gene expression in prokaryotes

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1 genetranscription

1 RNA

degraded 1 tRNA etc.

RNA processing

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

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operontwo or more genes transcribed together

a single RNA molecule that represents more than one gene

polycistronic message

2. Expression of operons

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A B CDNA

transcription

polycistronicmRNA

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a. Operons can encode several polypeptides or proteins.

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1 operonA B C

2 or more polypeptides

translation

AB

C

1 polycistronic mRNA

transcription

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1 operon

processing

rRNArRNA

degraded2 or more rRNAs

b. Operons can encode several rRNA molecules.

1 polycistronic RNA

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3. Important points

Most prokaryotes use operons.Operons are used to coordinategene expression and often containgenes of related function.

The details of organization, processing and degradation are different for different RNAs.

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

1. Expression of eukaryotic rRNA and tRNA genes

D. Eukaryotic gene expression

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geneE I I IE E E

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

2. Eukaryotic protein expressiona. Typical eukaryotic genes have exons and introns.

Eukaryotes do NOT have operons TB

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1 gene with exons and intronsE I I IE E E

transcription

1 RNA representing exons and introns(primary transcript)

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primary transcript

1 polypeptide

1 mRNA

processing

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b. Primary transcripts

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c. Processing of primary transcripts

i. cappingii. splicingiii. tailing

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i. CappingAddition of a 5' cap

CAP

Capping usually occurs beforetranscription is finished.

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OCH2

HO

N

N N

N NH2

O

OH

CH3Typical 5' CAP

7-methylguanosine

PP

P

5' carbonof RNA chain

5' to 5' linkage

O

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

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ii. SplicingThe removal of introns.

primary transcriptsplicing

RNA without intronsTB

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Addition of a poly-A tail

iii. Tailing

A1A2...A~200

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3. Notes on eukaryotic RNA processing

Processing occurs in the nucleus

The exact order of capping, tailing and splicing varies for different genes.

Poly-A tails are added by poly-Apolymerase, NOT during transcription.

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TRANSLATION

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III Prokaryotic translation

1. Key components of translation 2. Steps in translation3. The genetic code

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Overview of prokaryotic translationProtein synthesis from an mRNA template.

mRNA

translated region

translation

protein of specific amino acid sequenceTB

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1. Key components of translation

A. mRNAB. tRNAC. ribosomes and rRNA

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translated regionseries of codons

(usually ~300 codons)

mRNA

start codon

A. mRNA

stop codon

Shine-Dalgarno sequence

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RNA template for protein synthesis

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1. Shine-Dalgarno sequence~AGGAGG, ribosome binding sequence, critical for ribosome binding

2. start codonsAUG, GUG, or UUG

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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. TB

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AUGCAUUGUUCU...codons

protein fMet1 2

- His3

- Cys4

- Ser ...

5. Codons consist of 3 bases

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1 2 3 4

startcodon

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B. tRNAThe adapter molecule for translation

1. Particular tRNAs carry particular amino acids.

TBtRNA-f-Met

f-Met

tRNA-His

His

His

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AUGCAUUGUUCU...

codons

AA1 AA2

tRNAs

2. Particular tRNAs recognize particular codons.

amino acid (AA)

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

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3. tRNA structureAll tRNAs are generally similar in structure.

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a. 1o structure

ssRNA 73-93 nucleotides long

5' 3'UAC

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b. 2o structureclover leaf

anticodon loop

TC armD-arm

acceptor arm

extra arm

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c. 3o structure inverted "L"

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

anticodonBase pairing between an anticodon and a codon allows a tRNA to recognize a specific codon. TB

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e. codon-anticodon interactions

AAU5' 3' mRNA

codon1 2 3

UUA

anticodon123

5'3'

tRNA

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4. tRNA charging (adding amino acid)

3'

tRNA(uncharged)

3'H2N-CH-C-OR

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aminoacyl-tRNA(charged)

tRNA charging uses the energy of ATP TB

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Aminoacyl-tRNA synthetases

amino acidATP

tRNAaminoacyl-AMP

AMP PPiaminoacyl-tRNA

AMP = adenosine monophosphate PPi = inorganic pyrophosphate

enzymes that attach amino acids to tRNA

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enzyme

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5. tRNA facts

tRNAs contain many modified bases.

Prokaryotes have about 60 differenttRNAs.

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C. Ribosomes and rRNA

Ribosomesribonucleoprotein complexes that

catalyze protein synthesis.

rRNAs have structural and catalytic roles

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1. Prokaryotic 70s ribosome

23s rRNA5s rRNA34 proteins

16s RNA21 proteins

50ssubunit

30ssubunit

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A

2. Ribosomal sites where tRNAs bind

E = exit

PP = peptidyl

A = aminoacyl

E

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3. 16S rRNA

The 3' end of the 16s rRNA is complementary to the Shine-Dalgarno sequence (ribosome binding sequence of mRNAs)

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AUG

P-site f-met

Shine-Dalgarno(AGGAGG on mRNA)

II. Steps in translation

mRNA

A. initiation 30s subunitof ribosome

50s subunit

GTP hydrolysis

f-met

30s subunit TB

AGGAGG-----AUG

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

In Bacteria, different met-tRNAs are used forelongation and initiation.

initiation, formyl-methioninetRNAmetf

elongation, methioninetRNAmetm

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In Eukarya, the ribosome recognizes the 7-methylguanosine cap at the 5’ end ofmRNA and initiates at the first AUG.

In Eukarya and Archaea, initiation begins with methionine rather than f-met.

In Bacteria, the formyl group of the initiator formylmethionine (f-met) is later removed.

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2. Initiation in different domains

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AA

P-site A-site

AA1. AA-tRNA binding

AA AA

mRNA

B. Elongation

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AA AA

AAAA

(peptidyl transferase)

2. peptide bond synthesis

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3. translocation

AAAA

GTPhydrolysis

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AAAA

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AAAA

C. Termination

AAAAAA

UAA

AAAA

AAAAAA

termination

stop codon

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"Polysomes" are mRNAs with several ribosomes attached.

mRNA

mRNAs can be translated by 5-10 ribosomes simultaneously.

1. Ribosomes move along the mRNA.

D. Additional notes on translation

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2. In prokaryotes only, transcription and translation are coupled.

Translation begins before transcription ends.

DNA

mRNA TB

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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

properly folded protein

improperly folded protein molecular

chaperone

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III. The genetic code

A. universal codeB. degenerate code

1. synonyms2. codon families3. 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 memorize)

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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

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UUU phenylalanineUUC phenylalanine

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1. synonymscodons that code for the sameamino acid

Not all synonyms are used with equal frequency. This is called "codon usage bias".

UUU phenylalanineUUC phenylalanine

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2. codon families

CUUCUCCUACUG

leucine

any nucleotide in the 3rd positions

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3. codon pairs

UUUUUC phenylalanine

any pyrimidine in the 3rd position

CAACAG glutamine

any purine inthe 3rd position

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C. Wobble base pairing

UUUAAG

codon (mRNA)

anticodon (tRNA)

5'3'

3'5'

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

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Regulation ofGene Expression

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Note: put allosteric regulation in protein / enzyme section

Regulation of Gene Expression I:I. Regulation of gene expressionII. Transcriptional regulationIII. Examples of gene repressionIV. Example of gene induction

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Not all genes are turned on (expressed) all the time

In general, they are turned ononly when needed.

I. Regulation of gene expression

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Cells can respond to environmental changes by regulating gene expression.

glucose

maltose

lactose

arginine

tryptophan

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Different genes are expressed when cells grow on different compounds.

maltoseglucose

TCA

lactose

P O lacZ lacY lacA

lac permease (transport protein)-galactosidase

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

(galactose--1,4-glucose)

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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. constitutive2. regulated

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1. Constitutive gene expression

e.g. "housekeeping genes" like primase ssDNA binding proteins

expression of genes at about the same level under all environmental conditions

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2. Regulated gene expressionControl 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

amount of gene product

time

inducer

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enzymes for tryptophanbiosynthesis(molecules/cell)

time

tryptophan absent tryptophan present

e.g. genes that encode enzymes for tryptophan biosynthesis are repressed by tryptophan.

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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 proteinsB. Regulatory protein binding sitesC. Effector molecules

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A. Regulatory proteins

• Cells have many different regulatory proteins.• Specific regulatory proteins control the transcription of specific groups of genes.

• Transcriptional regulation is mediated by regulatory proteins.

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• Examples of regulatory proteins are "repressor proteins" and "activator proteins."

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DNA

RNA polymeraseP

Promoter

Repressor protein (dimer)

Repressor proteins decrease transcription when bound to DNA by interfering with the activity or binding of RNA polymerase.

1. Repressor proteins

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RNA polymerase

2. Activator proteins

DNA

P

"weak" promoter

Activator protein

Activator proteins increase transcription when bound to DNA by helping RNA polymerase bind to weak promoters. TB

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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

Imperfect palindrome

GTGTAAACGATTCCAC

CACATTTGCTAAGGTG

binding sites for repressor proteins

Usually found near promoters.

lac repressor binding site

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2. Activator binding sites

GTGAGTTAGCTCAC

CACTCAATCGAGTG

Imperfect palindrome

Binding sites for activator proteins

Usually found near promoters.

crp binding

site

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

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e.g. catabolic substrates: sugars, amino acids, fatty acids

a. inducerssmall molecules that mediate gene induction

1. Classes of effectors

lactose

maltose

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e.g. biosynthetic products:amino acids, purines, pyrimidines, fatty acids etc.

b. corepressorssmall molecules that mediate gene repression

tryptophanarginineTB

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2. How effectors work

conformational change (change in 3-D structure)

regulatory protein effector

Effectors change the DNA binding affinityof regulatory proteins for their binding sites.

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DNA

conformational change(change in 3-D structure)

regulatory protein

effector

A. Some effectors increase DNA binding affinity

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DNA

regulatory protein

conformational change(change in 3-D structure)

B. Some effectors decrease DNA binding affinity

effector TB

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Since most regulatory proteins influence transcription when bound to DNA, the binding of effectors to regulatory proteins changes gene expression.

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effector

regulatory protein

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III. Examples of gene repression

A. Regulation of the trp operonB. Regulation of the arg operon

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The trp operon

polycistronic mRNA

E D C B A

Five enzymes for tryptophan biosynthesis

trp genespromoter

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

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2. When Trp is available, E. coli takes up Trp from the environment and represses the trp operon.

1. When Trp is NOT available in the environment, expression of the trp operon allows Escherchia coli to make Trp needed for protein synthesis.

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trp promoter

operatorinactiverepressor

genes on TB

RNA polymerase

tryptophan

activerepressor

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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P

B. Regulation of the arg operon for arginine biosynthesis

operator arg biosynthetic genesargC argB argH

If arginine is present in large amounts • arg biosynthetic enzymes NOT needed

• RNA polymerase can't bind to promoter

• arg binds repressor • arg-repressor binds DNA

transcription rate decreases

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Poperator arg biosynthetic genes

argC argB argH

If arg is absent, the cell needs to make arg • repressor doesn't bind DNA

• RNA polymerase can bind • transcription of arg genes occurs

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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.

Z Y A

lac genespromoter

operatorTB

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

• When lactose is unavailable, the catabolic enzymes are NOT needed.

The lac operon isexpressed at only very low levels.

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B. Lactose unavailable

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

Z Y A

lac promoter

genesoff

Note: the role of crp/cAMP in control of thelac operon is not considered here. TB

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Z Y A

C. Lactose available lac promoter

geneson

RNA polymerase

lactose allolactose

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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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

P O lacZ lacY lacACAPsite

Some repressor proteins mediate gene induction.Example: the lac repressor

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P O lacZ lacY lacA

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

CAPsite

Some repressor proteins mediate gene induction.

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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/CAPcomplex

binds DNA

D. The catabolite activator protein

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O

N

N N

N

NH2

CH2

OH

HOP=O

O

cyclic AMP (cAMP)cyclic adenosine monophosphate

(Don't memorize)

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P O lacZ lacY lacAcrp

P O lacZ lacY lacAcrpbinding site

Without activator protein, RNA polymerase binds weakly and the transcription rate is low.

With activator protein (crp), RNA polymerasebinds well and the transcription rate is higher.

Role of CAP (crp) in the lac operon

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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).

bacterial chromosome

crp binding site

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1. Catabolite repression enables Escherichia coli to use glucose in preference to other carbon sources.

maltoseglucose

TCA

P O lacZ lacY lacAcrpbinding site

lac permease (transport protein)

lactose

-galactosidase

Lactose utilization requires additional proteins.

(galactose--1,4-glucose)

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a. cAMP (cyclic AMP)an effector molecule that increases the DNA binding affinity of the catabolite activator protein

b. CAP (or crp) Catabolite activator protein, a transcriptional regulatory protein; also called crp (cAMP receptor protein)

2. Key components of catabolite repression

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CAP (or crp)

bacterialchromosome

CAP (or crp) binding sites

cAMP

cAMP/CAPcomplex

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

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c. Without cAMP/CAP, genes required to catabolize nonglucose energy sources are transcribed at very low rates.

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.

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 operonrequires both lactose andcAMP/CAP for induction.

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P O lacZ lacY lacAcrpbinding site

Both lactose and cAMP/CAP are needed for high induction of lac operon.

P O lacZ lacY lacAcrp

glucose absent, lactose present

glucose present, lactose absent

lac repressor binds DNA in absence of lactose

glucose decreases cAMP

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III. Two-component regulatory systems

Transcriptional regulatory systems composed of a sensor kinase andresponse regulator.

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A. Sensor kinaseIntegral membrane proteins thatsense environmental conditions andphosphorylate proteins

B. Response regulator

Cytoplasmic transcriptional regulatoryproteins controlled by sensor kinasesthrough phosphorylation TB

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cytoplasmic membrane

sensorkinase

effector

P

response regulator

PPdephosphorylation

phosphorylation

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