MCB 3020, Spring 2005 Chapter 7: Molecular Genetics

1

MCB 3020, Spring 2005

Chapter 7:Molecular Genetics

2Molecular Genetics I: ReplicationI. Heredity and GeneticsII. GenomesIII. DNA structureIV. Bacterial DNA replicationV. Replication at the ends of linear DNA

3I. HeredityThe 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

4

the study of the mechanisms of heredity and variation in organisms

Genetics:

TB

5

Flow of information replication DNA DNAtranscription RNAtranslation protein

6

Genome = all the DNA of a cell (or all the genetic

material of a virus)

II. Genome

7

one circular double-stranded DNA chromosome

oftenplasmid(s)

Typical bacterial genome

500-12,000 genes TB

8typical viral genome

DNAor

RNA

4-200 genes TB

9Typical eukaryotic genome

4-224, linear chromosomes

5,000 - 125,000 genes TB

10III. DNA structuredeoxyneucleotidesphosphodiester bonds5' and 3' endsantiparallelcomplementarydouble helix

TB

11

N

N N

N

NH2

Deoxyadenosine (purine)

HOCH2

TB

HO H

12

HO

NH

N

deoxythymidine (pyrimidine)

O

OHOCH2

TB

H

H3C

13

O

-P-O-C

OP

OC

-O

O-

phosphodiester bond

ssDNATB

14

O

-P-O-C

OP

OC

HO

O-

3’ end ssDNA

5' end

5’

2’

1’

3’

4’

ring numbering system for

deoxyribose

-C

TB

15dsDNA

5’5’3’3’

antiparallel

dsDNA is always antiparallel

TB

16complementary

GGATGCGT

3’-CCTACGCA-5’

Two ssDNA molecules joined bystandard base-pairing rulesIn dsDNA, the strands are alwayscomplementary.

TB

5’- -3’

17

double helix

right handed

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18Supercoiling

relaxed DNA supercoiled DNA

TB

Within cells DNA is supercoiled

19IV. Bacterial DNA replication

DNA synthesis using a DNA template

Complementary base pairing (A=T, GC) determines the sequence of the newly synthesized strand.

DNA replication always proceeds from 5’ to 3’ end.

TB

20


21Overview of bacterial DNA replication

single origin (in bacteria)bidirectionaltheta structuresreplication forksemi-conservative

TB

22bacterial DNA replication

bacterialchromosome

origin (start point) bidirectional

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23

two replication forks

thetastructure

TB

24semi-conservative

+

**

*

* TB

25

Key EnzymeshelicasessDNA binding proteinprimaseDNA polymerase IIIDNA polymerase IDNA ligase

TB

IV. Bacterial DNA replication

26

All DNA polymerases require a primer

DNA is synthesized 5' to 3'

Important facts

TB

27helicase

Unwinds duplex DNA

TB

28ssDNA binding protein

binds to and stabilizes ssDNA

prevents base pairing

ssDNA binding proteinTB

29primase

synthesizes a short RNA primerusing a DNA template

RNA primer(a short starting sequence made of RNA)

primase

TB

30DNA polymerase III

Synthesizes DNA from a DNAtemplate and proofreads

TB

31DNA polymerase I

Synthesizes DNA from a DNAtemplate and removes RNA primers.

TB

32DNA ligaseJoins DNA strands together by forming phosphodiester bonds

DNA ligase

TB

33replication fork

5'

5'

3'

3'

template strands

lagging strand

leading strand

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34

helicasessDNA binding proteins

RNA primer

3'

5'Leading strand synthesis

TB

35

helicase

ssDNA binding proteins

DNA polymerase

3'

5'

TB

36

helicase

ssDNA binding proteins

DNA pol III

3'

5'Leading strand synthesis

TBDNA

37

helicasessDNA binding proteins

(primase)pol III

3'

Lagging strand synthesis (discontinuous) Okazaki

fragment(~1000 bases)

TB

3'

5'

38Primer removalpol III

pol I

pol I

3'

5'

5’ to 3’exonucleaseactivity

TB

39

DNA ligase

Ligation

TB

40Proofreading

Pol III removes misincorporated basesusing 3' to 5' exonuclease activity

This decreases the error rate to about10-10 per base pair inserted

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41

Since all known DNA polymerasesneed a primer, how are the ends oflinear DNA replicated in eukaryotes?

5' 3'

RNA primer

template

newly synthesized DNA

TB

V. Replication of the ends of linear DNA

42

repetitive DNA at the end of lineareukaryotic chromosomes

Telomeres

(GGGGTT)n

Example

n = 20 - 200

GGGGTT GGGGTT GGGGTT

5' TB

43Telomerases are enzymes that add DNA repeats to the 3' end of DNA.

Telomerases are composed of protein and an RNA molecule that functions as the template for telomere synthesis.

AACCCCAAC

telomerase

44

AACCCCAAC

5'GGGGTTGGGGTT

5'

telomerase

45

AACCCCAAC

5'

5'GGGGTTGGGGTT GGGGTT

primase

GGGGTT GGGGTT GGGGTT

46pol III

pol I5'

ligase

telomeric repeats

47For most cells, telomeres are added during development. Later telomerase becomes inactive.

Hence, as cells divide the DNA becomes shorter.

TB

Note that telomerase is reactivated in many types of cancer cells.

48Study objectives1. Compare and contrast bacterial, viral and eukaryotic genomes.2. What are the 4 bases in DNA? Which are purines? Which are pyrimidines? What is the sugar? I will not ask you to recognize the structures of individual bases, but note that deoxythymidine has a methyl group in the pyrimidine ring.3. Understand how the following terms apply to DNA structure: phosphodiester bonds, 5' and 3' ends, antiparallel, complementary, double helix. What parts of the nucleotides are joined in the phosphodiester bond? 4. Understand how the following terms apply to DNA replication: template, complementary base pairing, origin, bi-directional, theta structures, replication fork, semi-conservative.5. Know how the following enzymes function in leading and lagging strand replication: helicase, ssDNA binding protein, primase, DNA polymerase III, DNA polymerase I. What is an Okazaki fragment?6. What is proofreading?7. Understand the problem of replicating the ends of linear DNA. Understand how telomerase solves that problem for eukaryotic chromosomes.

49Molecular Genetics II: TranscriptionI. RNA II. Gene expressionIII. Prokaryotic Transcription

50


51I. 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.

TB

52A. 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

phe

53B. RNA structure

1. RNA nucleosides2. phosphodiester bonds3. 5' and 3' ends4. complementary base pairing5. stem-loops

TB

54

The RNA nucleosides have 2'-hydroxyl groups which arenot found in DNA.

1. The RNA nucleosides

"U" is found in RNA (in place of "T")

Guanosine (G)Adenosine (A)

Cytidine (C)Uridine (U) TB

55

2. The phosphodiester bonds of RNA are analogous to those of DNA.

3. The 5' and 3' ends of RNA are analogous to those of DNA.

TB

56

O

P-O-C5' end

ring numbering system for ribose

O

5'

2’

1’

3’

4’-C O

OP

OC

HO

O-

3’ end RNA

O

OH

OHphosphodiester

bond

TB

574. Complementary base pairing

CCCUUUGGGAAA

GGGAAACCCUUU RNA

RNA

GGGAAACCCUUU RNA

CCCTTTGGGAAA DNA

TB

hydrogenbonding

585. RNA stem loops

complementarybase pairing(helical)ssRNA

A common RNA secondary structure

TB

59II. Gene expressionDifferent scientists define the termgene expression differently. Most commonly, gene expression refers tothe decoding of genes into proteinsor RNAs.

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

60A. Gene numbers

virusesprokaryotes eukaryotes

groupapproximategene number

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

TB

61Any 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

62

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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63

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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64

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

65a. 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

66

1 operon

processing

rRNArRNA

degraded2 or more rRNAs

b. Operons can encode several rRNA molecules.

1 polycistronic RNA

TB

673. 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.

TB

68

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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69

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

701 gene with exons and intronsE I I IE E E

transcription

1 RNA representing exons and introns(primary transcript)

TB

71

primary transcript

1 polypeptide

1 mRNA

processing

TB

b. Primary transcripts

72c. Processing of primary transcripts

i. cappingii. splicingiii. tailing

TB

73i. CappingAddition of a 5' cap

CAP

Capping usually occurs beforetranscription is finished.

TB

74

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.

75ii. SplicingThe removal of introns.

primary transcriptsplicing

RNA without intronsTB

76

Addition of a poly-A tail

iii. Tailing

A1A2...A~200

TB

773. 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.

TB

78E. Comparison of eukaryotic and prokaryotic gene expression.

Eukaryotic mRNAs are usuallyspliced,capped and tailed.

Eukaryotes do NOT have operons.

tRNA and rRNA expression are generally similar

Prokaryotic genes very very rarely have introns TB

79III. Prokaryotic transcription

A. overviewB. transcribed regionsC. RNA polymeraseD. promotersE. terminatorsF. sigma factor

TB

80


81A. Overview of prokaryotic transcription

RNA polymerase

primary transcript complementary to one strand of the coding region

RNA synthesis from a DNA template

typicalgene dsDNA

TB

82B. Defined regions are transcribed

upstream region

transcribedregion

downstream region

promoter(RNA polymerase

binding site)

transcriptionstart site

terminationsite

gene dsDNA

TB

83

gene,or operon

RNA polymerase

C. RNA polymerase is the enzyme that synthesizes RNA from a DNA template.

complementary RNA

DNA template

TB

84

++ completed

transcriptTB

85D. PromotersSites on DNA where RNA polymerase binds to start transcription

promoter

upstream region

transcribedregion

downstream region

transcriptionstart site

terminationsite

gene dsDNA

TB

861. Typical bacterial 70 promoter

TTGACA TATAATAACTGT ATATTA

TATAAT = -10 consensus

sequence

TTGACA = -35 consensus

sequence

TB

*also called Pribnow box; ~ 10 bases before start

site of transcription

87A more common way to draw a promoter

TTGACA TTAACT

-10-35

Note:The - 10 and -35 sequences can vary somewhat.

5' 3'

TB

88E. Transcriptional terminatorsDNA region that mediates the termination of transcription.

gene dsDNA

region whereterminators areusually found

terminationsite

TB

891. Intrinsic terminator

DNA encoding an RNA that formsa stem loop followed by a run of "U"sthat is used for transcriptional termination.

UUUURNA

3' end of RNATB

90

The RNA stem loop binds to RNA pol and causes termination

Intrinsic terminator function

Important fact: Intrinsic terminators must betranscribed in order to function. TB

912. Rho-dependent terminatorA DNA site where RNA polymerasepauses and transcription is terminated by Rho protein

TB

92

Rho protein

Rho protein binds RNA then moves along RNA until it contacts RNA pol and terminates transcription

RNA polpauses at

Rho termination siteTB

93F. The sigma factor cycle

Sigma factors are needed for promoter binding, but after transcription starts they dissociate.

Sigma factors ( ) are a subunit of RNA polymerase.

TB

941. Subunit structure of bacterial RNA polymerase

'

core enzyme

The holoenzyme includes one of several sigma factors. TB

'

holoenzyme

95RNA pol holoenzyme (core + sigma)

sigmafactor

RNA (~10 nucleotides)

sigma factor

core enzyme

TB

96

+core enzyme

termination

RNA

sigma

holoenzyme TB

97

TAATGTGAGTTAGCTCACTCATTA

GGCACCCCAGGCTTGACATTTATG

CTTCCGGCTCGTATGTTGTGTGGA

AATTGTGAGCGGATAACAATTTCA

CACAGGAAAGAGCTATGACC...

Upstream region of the lactose operon

-35 region

-10 region (Pribnow)

Shine-dalgarno (RBS)

Translation start site

Transcription start site

98Study objectivesYou will need to know ALL the concepts and details in this lecture.1. What are the three main types of RNA and what are their functions?2. Understand how the following terms apply to RNA structure: phosphodiester bonds, 5' and 3 ends, nucleosides, complementary base pairing, stem loops.3. Compare and contrast DNA and RNA structure.4. What is a gene? What is gene expression? *Understand transcription, translation, and RNA processing in both prokaryotes and eukaryotes. 5. Define operons and polycistronic messages. How do they function in prokaryotic gene expression? 6. *Compare and contrast the features of prokaryotic and eukaryotic gene expression. Do eukaryotes have operons? What are exons, introns, primary transcripts, capping, tailing, and splicing. What is the 5' cap (methylguanosine cap)? How and when is the poly-A tail added to the transcript? Where does eukaryotic RNA processing occur? 7. Understand the structure and function of promoters and terminators in transcription. Contrast intrinsic terminators and rho-dependent terminators.8. Know the subunit structure of bacterial RNA polymerase and the sigma cycle.

99Molecular Genetics III: Prokaryotic translation

I. Key components of translation II. Steps in translationIII. The genetic code

100Overview of prokaryotic translationProtein synthesis from an mRNA template.

mRNA

translated region

translation

protein of specific amino acid sequenceTB

phe

101I. Key components of translation

A. mRNAB. tRNAC. ribosomes and rRNA

102

translated regionseries of codons

(usually ~300 codons)

mRNA

start codon

A. mRNA

stop codon

Shine-Dalgarno sequence

TB

RNA template for protein synthesis

1031. Shine-Dalgarno sequence~AGGAGG, ribosome binding sequence, critical for ribosome binding

2. start codonsAUG, GUG, or UUG

TB

3. stop codons (nonsense codons)

UAA, UGA, or UAG

1044. 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

105

AUGCAUUGUUCU...codons

protein fMet1 2

- His3

- Cys4

- Ser ...

5. Codons consist of 3 bases

TB

1 2 3 4

startcodon

106B. tRNAThe adapter molecule for translation

1. Particular tRNAs carry particular amino acids.

TBtRNA-f-Met

f-Met

tRNA-His

His

His

107

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

1083. tRNA structureAll tRNAs are generally similar in structure.

TB

a. 1o structure

ssRNA 73-93 nucleotides long

5' 3'UAC

109b. 2o structureclover leaf

anticodon loop

TC armD-arm

acceptor arm

extra arm

TB

110c. 3o structure inverted "L"

TB

111d. 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

112e. codon-anticodon interactions

AAU5' 3' mRNA

codon1 2 3

UUA

anticodon123

5'3'

tRNA

TB

1134. tRNA charging (adding amino acid)

3'

tRNA(uncharged)

3'H2N-CH-C-OR

O

aminoacyl-tRNA(charged)

tRNA charging uses the energy of ATP TB

114Aminoacyl-tRNA synthetases

amino acidATP

tRNAaminoacyl-AMP

AMP PPiaminoacyl-tRNA

AMP = adenosine monophosphate PPi = inorganic pyrophosphate

enzymes that attach amino acids to tRNA

TB

enzyme

1155. tRNA facts

tRNAs contain many modified bases.

Prokaryotes have about 60 differenttRNAs.

TB

116C. Ribosomes and rRNA

Ribosomesribonucleoprotein complexes that

catalyze protein synthesis.

rRNAs have structural and catalytic roles

TB

1171. Prokaryotic 70s ribosome

23s rRNA5s rRNA34 proteins

16s RNA21 proteins

50ssubunit

30ssubunit

TB

118

A

2. Ribosomal sites where tRNAs bind

E = exit

PP = peptidyl

A = aminoacyl

E

TB

1193. 16S rRNA

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

120

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

1211. f-met tRNA (formyl-methionine tRNA)

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

initiation, formyl-methioninetRNAmetf

elongation, methioninetRNAmetm

TB

122

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.

TB

2. Initiation in different domains

123

AA

P-site A-site

AA1. AA-tRNA binding

AA AA

mRNA

B. Elongation

TB

124

AA AA

AAAA

(peptidyl transferase)

2. peptide bond synthesis

TB

1253. translocation

AAAA

GTPhydrolysis

TB

AAAA

126

AAAA

C. Termination

AAAAAA

UAA

AAAA

AAAAAA

termination

stop codon

TB

127

"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

TB

1282. In prokaryotes only, transcription and translation are coupled.

Translation begins before transcription ends.

DNA

mRNA TB

1293. 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

130III. The genetic code

A. universal codeB. degenerate code

1. synonyms2. codon families3. codon pairs

C. wobble base pairing

131III. The genetic code 8 codon families, 14 codon pairs, 3 stop codons

(Do not memorize)

132A. The genetic code is almost universal.

Most organisms use the same genetic code.

TB

133B. The genetic code is degenerate.

more than one codon can code for the same amino acid

TB

UUU phenylalanineUUC phenylalanine

1341. synonymscodons that code for the sameamino acid

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

UUU phenylalanineUUC phenylalanine

1352. codon families

CUUCUCCUACUG

leucine

any nucleotide in the 3rd positions

TB

1363. codon pairs

UUUUUC phenylalanine

any pyrimidine in the 3rd position

CAACAG glutamine

any purine inthe 3rd position

TB

137C. 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.

TB

138


139Study objectives1. Know the DETAILS of the structure and function of mRNA, tRNA, rRNA, and ribosomes in translation. Memorize the start and stop codons. You do NOT need to memorize codons other than the start and stop codons. 2. What reaction is catalyzed by aminoacyl-tRNA synthetases? 3. For the process of translation, know the details of initiation, elongation peptide bond formation, translocation and termination. 4. Compare and contrast Bacterial, Archaeal and Eukaryal initiation.5. What are polysomes?6. What is meant when it is said that transcription and translation are coupled in prokaryotes? 7. Some proteins fold spontaneously while others require assistance. What are molecular chaperones?8. How do the following terms apply to the genetic code: synonyms, codon pairs, codon families, wobble, codon usage bias.

140


Chapter 7:Regulation of

Gene Expression

141

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

142

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

In general, they are turned ononly when needed.

I. Regulation of gene expression

TB

143Cells can respond to environmental changes by regulating gene expression.

glucose

maltose

lactose

arginine

tryptophan

144Different 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)

145A. Why regulate gene expression?

Regulation allows cells to respond to environmental conditions by synthesizing selected gene products only when they are needed.

146B. Gene expression synthesis of a gene product

1. constitutive2. regulated

1471. Constitutive gene expression

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

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

TB

1482. 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

149a. gene induction increase in gene expression

amount of gene product

time

inducer

TB

150

maltosecatabolicenzymes(molecules/cell)

time

maltose absent

e.g. genes that encode maltose-utilizing enzymes are induced by maltose.

maltose added

lag phase

151b. gene repression decrease in gene expression

amount of gene product

timeTB

152

enzymes for tryptophanbiosynthesis(molecules/cell)

time

tryptophan absent tryptophan present

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

153Important 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

154II. 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

TB

155A. 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.

TB

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

156

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

TB

157

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

158B. 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.

TB

1591. Operator sites

Imperfect palindrome

GTGTAAACGATTCCAC

CACATTTGCTAAGGTG

binding sites for repressor proteins

Usually found near promoters.

lac repressor binding site

TB

1602. Activator binding sites

GTGAGTTAGCTCAC

CACTCAATCGAGTG


Binding sites for activator proteins

Usually found near promoters.

crp binding

site

TB

161C. Effector molecules

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

TB

162

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

a. inducerssmall molecules that mediate gene induction

1. Classes of effectors

lactose

maltose

TB

163

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

b. corepressorssmall molecules that mediate gene repression

tryptophanarginineTB

1642. 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.

TB

165

DNA

conformational change(change in 3-D structure)

regulatory protein

effector

A. Some effectors increase DNA binding affinity

TB

166

DNA

regulatory protein

conformational change(change in 3-D structure)

B. Some effectors decrease DNA binding affinity

effector TB

167Since most regulatory proteins influence transcription when bound to DNA, the binding of effectors to regulatory proteins changes gene expression.

TB

effector

regulatory protein

168

TB

III. Examples of gene repression

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

169

The trp operon

polycistronic mRNA

E D C B A

Five enzymes for tryptophan biosynthesis

trp genespromoter

TB

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

170

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.

TB

171trp promoter

operatorinactiverepressor

genes on TB

RNA polymerase

tryptophan

activerepressor

genes off

172Note: 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 .

173

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

174

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

175IV. 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

176

• 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.

TB

177B. 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

178

Z Y A

C. Lactose available lac promoter

geneson

RNA polymerase

lactose allolactose

repressor does not bind DNA TB

179Important 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.

TB

180

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

181

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.

+

182Study objectives: Please study all the concepts and details of Regulation.1. Why do cells control gene expression? What is constitutive gene expression?2. What is gene induction? gene repression?3. Are catabolic genes more likely to be repressed or induced? Why?4. Are anabolic (biosynthetic) genes more likely to be repressed or induced? Why?5. What are the functions of the following in the regulation of transcription: repressor protein, activator protein, effector, co-repressor, inducer, activator binding site, operator, palindromic sequences, protein conformational changes? Understand the concepts and details. Do NOT memorize the specific palindromic sequences.6. Describe regulation of the trp operon and arg operon by repressor proteins.7. Describe the effect of lactose on the induction of the lac operon.8. Explain how repressor proteins can mediate gene repression. Explain how repressor proteins can mediate gene induction.9. Know that some activator proteins can mediate gene induction, while other activator proteins mediate gene repression

183Regulation of Gene Expression II:I. Activator proteinsII. Global regulationIII. Two-component regulatory systemsIV. Attenuation

184I. Activator proteins

eg. maltose activator proteincatabolite activator protein (CAP) cyclic AMP receptor protein (crp)

Proteins that activate transcription when bound to activator binding sites.

185

RNA polymerase

A. Typical activator protein

DNA

activator binding site

P

unusual promoter

TB

186B. Typical activator binding site


GTGAGTTAGCTCACCACTCAATCGAGTG

P O

TB

187C. Unusual promoters are involved in control by activator proteins.

-10consensus

(Pribnow box)

No-35

consensus

TB

188

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

TB

189

O

N

N N

N

NH2

CH2

OH

HOP=O

O

cyclic AMP (cAMP)cyclic adenosine monophosphate

(Don't memorize)

TB

190

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

1913. 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

192

Bacterial chromosome

operator oractivator binding sitesof similar DNAsequence

II. Global regulation A. Control of many genes by a single regulatory protein

TB

193B. Example: catabolite repressionA global regulatory system that allows glucose to be consumed in preference to a variety of other carbon sources.

TB

1941. Catabolite repression enables Escherichia coli to use glucose in preference to other carbon sources.

maltoseglucose

TCA


lac permease (transport protein)

lactose

-galactosidase

Lactose utilization requires additional proteins.

(galactose--1,4-glucose)

195

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

TB

196

CAP (or crp)

bacterialchromosome

CAP (or crp) binding sites

cAMP

cAMP/CAPcomplex

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

TB

197

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.

198C. Global regulation is often used together with other more specific regulatory systems.

Example: the lactose operonrequires both lactose andcAMP/CAP for induction.

199


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

200III. Two-component regulatory systems

Transcriptional regulatory systems composed of a sensor kinase andresponse regulator.

TB

201A. Sensor kinaseIntegral membrane proteins thatsense environmental conditions andphosphorylate proteins

B. Response regulator

Cytoplasmic transcriptional regulatoryproteins controlled by sensor kinasesthrough phosphorylation TB

202

cytoplasmic membrane

sensorkinase

effector

P

response regulator

PPdephosphorylation

phosphorylation

TB

203

Phosphorylation changes the DNA binding affinity of the response regulator.

C. Transcriptional control

When response regulators are bound to DNA, they induce or repress gene expression.

TB

204IV. Attenuation

A "fine tuning" system for regulating gene expression by control of transcriptional termination.

1 2 3 4UUUUU

conformation 2 intrinsictranscriptional

terminator

TB

205A. The trp operon is regulated at two levels.

P O E D C B A

1. repression by trp repressor (on/off)

genes encoding the enzymes used for tryptophan biosynthesis

2. attenuation (fine tuning by transcriptional termination)

R

R

206B. Leader region and leader peptide

P O E D C B A

leaderregion

tryptophan biosynthesis genes "structural genes"

tryptophan biosynthetic enzymesleader peptide

translation

transcription mRNA

207

leaderregion

coding region for trp enzymes

1. leader region of trp mRNAmRNA region upstream of the coding region for the trp biosynthesis enzymes

trp mRNATB

208

A short peptide encoded by the leader region of the trp mRNA.

trp leader mRNA (has 2 trp codons)

translation

leader peptide

2. leader peptide

met-lys-arg-ile-phe-val-leu-lys-gly-trp-trp-arg-thr-ser(Don't memorize sequence) TB

209

1. The leader region of the trp mRNA has four segments that can fold into 2 mutually exclusive conformations by complementary base pairing.

trp mRNA leader region

1 2 3 4

C. The trick to attenuation

TB

210mRNA leader region

1 2 3 4

conformation 1

2 31 4

1 2 3 4UUUUU

conformation 2 intrinsictranscriptional

terminator(3:4 loop) TB

2112. Conformation 2 of the trp mRNA leader is an intrinsic terminator.

Plentiful tryptophan favors conformation 2 and termination. Energy is not wasted making tryptophan when it is plentiful.

If conformation 2 is formed, transcription of the trp operon is terminated before the remainder of the trp mRNA is made.

1 2 3 4UUUUU

TB

212

1 2 3 42:3 stem loop mRNA

1 2 3 4UUUU

Intrinsicterminator(3:4 stem-loop)

3. The rate of TRANSLATION of the leader peptide determines which conformation (stem-loop) will form.

TB

213

a. When tryptophan is plentiful, translation of the trp leader peptide is FAST (i.e. ribosomes move fast).

4. The leader region encodes two tryptophans in a row.

Fast translation favors formation of the intrinsic terminator (the 3:4 loop). Transcription terminates before the structural genes are transcribed.

214b. When tryptophan levels are LOW, translation of the trp leader peptide is SLOW. The ribosome PAUSES at the trp codons, waiting for tryptophan-tRNA.

When the ribosome pauses, the 2:3stem-loop forms. The 3:4 intrinsic terminator stem-loop CANNOT form.Transcription of the trp biosynthesis genes continues.

215C. Transcription of the trp operon when tryptophan is plentiful.

1 Ribosome begins translation immediately after RNA synthesis occurs.

1 2 Ribosome finishes translation of the leader peptide and leavesthe mRNA.

21 Stem loop 1:2 forms. TB

2161 2 3 4 Transcription continues.

Stem loop 3:4 forms.

1 2 3 4UUUU

Stem loop 3:4 is an intrinsic terminator thatprevents furthertranscription.

UUUUU

P O E D C B A

leaderregion

tryptophan biosynthesis genes are NOT transcribed

X X X X X1 2 3 4

TB

217D. Transcription of the trp operon when tryptophan is low.

1 Ribosome begins translation immediately after RNA synthesis occurs.

1 2Because tryptophan is low, the ribosome pauses at tryptophancodons of the leader peptide and remains attached to the mRNA.

1 2 3Transcription continues.

TB

218

no terminator is formed

2 31The ribosome blocksbase pairing betweensegments 1 and 2.

42 31Segments 2 and 3 pairblocking the pairing of3 and 4.

Note that the alternative conformations of the trpleader mRNA are mutually exclusive. TB

219

no terminator is formed

P O E D C B A

tryptophan biosynthesis genes are transcribed

and translated

42 31

End result: When tryptophan levels are low, the genes for the tryptophan biosynthesis are expressed.

mRNA

DNA

proteins

220Study objectives: Please study both the concepts and details of Regulation.

1. What is an activator protein? How does it work? What is the catabolite activator protein or cAMP receptor protein (crp)? 2. What are the roles of cAMP, CAP and glucose in catabolite repression? 3. What is global regulation? Describe the example presented in class.4. How do the lac repressor system and cAMP/CAP system regulate expression of the lac operon? Understand (in detail) the effects of both lactose and glucose on the expression of the lac operon. 5. Describe how sensor kinases and response regulators function in two-component regulatory systems. 6. Understand the CONCEPTS and DETAILS of attenuation. What is the role of tryptophan, the leader peptide, the ribosome, and alternative leader mRNA conformations in trp operon attenuation?7. Compare and contrast (i) transcriptional regulation by regulatory proteins (ii) two-component regulatory systems and (iii) attenuation.

221


Chapter 8:Viruses

222Viruses:I. General properties of virusesII. Examples of virusesIII. Viral structureIV. Phage reproductionV. Reproduction of lysogenic phageVI. Overview of animal viruses

TB

223Typical viruses (30-200 nm)nucleic acid

helicalcapsid

envelope

icosahedral capsids

viralspecificproteins TB

224I. General properties of viruses A. small (~30-200 nm) B. non-cellular

TB

C. replicate within host cells and take over the host machinery D. released from the host cell and infect other cells virion = extracellular state of virus.E. often damage or kill the host

225

A. Human wart virusII. Some examples of viruses

Icosahedral symmetry (20 regular faces)TB

Picture18

226B. Tobacco mosaic virus

RNA virus

Helical symmetry

TB

Picture19

227C. Flu virus

enveloped virusTB

Picture20

228D. Lambda virus

host = a bacteriumbacterial viruses are also called

bacteriophage ("bacteria eaters") or phage

229III. Viral structure

A. genomesB. capsidsC. envelopesD. packaged enzymes

TB

230A. Viral genomes

All the hereditary material of a virus

dsDNAssDNAdsRNAssRNA

4 - 200 genes

TB

231B. Viral capsidsProtein shell that surrounds the genome

Protects the viral genomeOften needed for attachment to the host cellsUsually helical or icosahedral

capsid (protein coat)cross-section of

icosahedral capsidgenome

TB

232C. Viral envelopes

Composed of host lipids and viral proteins Often used for attachment to the host cell

lipids from host

viral proteins

TB

Outermost layer of enveloped viruses

233D. Packaged proteinsProteins found within the capsidDifferent functions in different viruses

viral proteine.g. reverse transcriptase RNA-dependent RNA polymerase TB

2341. Reverse transcriptase

enzyme that synthesizes DNA from an RNA template

2. RNA-dependent RNA polymerase

enzyme that synthesizes RNA froman RNA template

TB

235IV. Reproduction of phageA. AttachmentB. PenetrationC. Expression of viral genesD. Genome replicationE. Capsid formationF. PackagingG. Release

TB

236A. AttachmentBinding of a capsid or envelope protein to a host receptor.

host receptor

host cell

(usually a specific protein,lipid, or polysaccharide)

Specificity for the host receptordetermines virus host range TB

237Attachment and penetration

Virus tail fibers interacting with core polysaccharides

238B. penetration

injection ofviral nucleic acidand packaged

proteins.

TB

239C. Expression of viral genes

Viral proteins

viral genome

host machinery

TB

240

capsid proteinsproteins that block host gene expressionproteins that block restriction systems

Typical viral proteins

TB

proteins for genome replicationproteins for assembly of viral particles

241D. Genome replication

various methods: for example,host enzymes onlyviral enzymes onlyhost and viral enzymes

TB

242E. capsid formation self-assembly of capsid proteins

TB

243F. packaging

Insertion of the nucleic acid intothe capsid

Method varies

The "headfull" method is common

TB

244

1. Lysis G. Release

TB

2452. Budding (enveloped viruses)

viralproteins

host lipids

TB

246V. Reproduction of lysogenic phageA. lysisB. lysogenyC. prophage induction

TB

247A. LysisThe most frequent method of reproduction

Occurs as described above TB

248

bacterialchromosome

lysogen(cell with integrated virus)

integrationprophage

(integrated virus)

1. Prophage integration B. Lysogeny

TB

249

host replication

2. Prophage replication

TB

250C. Prophage induction

Excision of the prophagefollowed by lytic replication.

UV light and other DNA damaging agents cause prophage induction.

TB

251

binding to host receptor and uptake by endocytosis

1. Attachment and penetrationVI. Overview of animal viruses

animal celluncoatingTB

2522. Gene expression and genome replication for animal viruses must follow (or adapt to) eukaryotic rules

eukaryotic RNA processingcompartmentation (nucleus vs. cytoplasm)

TB

What features of transcription and translation would differ between phage and animal viruses?

253B. Host interactions

1. lysis2. persistent infection3. latent infection4. transformation

TB

2541. lysisdestruction of the host cell

2. persistent infectionviruses bud from host over a longperiod of time.

3. latent infectioninfections that reoccur periodically

4. transformationincreased growth rate of host cells TB

255Study objectivesPlease understand ALL the CONCEPTS and DETAILS presented in this lecture.

1. Describe the general properties of viruses.2. Define virions, bacteriophage, phage.3. Describe viral genomes, capsids (protein coats or shells), envelopes, and packaged proteins. What are the functions of these molecules? Know the specific examples presented in class.4. Compare and contrast the details of the reproductive cycles of phage and animal viruses. Thought question: What features of transcription and translation would differ between phage and animal viruses?5. How do phage reproduce by lysogeny?6. What is a lysogen? a prophage? 7. What effects can animal viruses have on their hosts?

256

I. Polio virusII. Flu virusIII. HIV virusIV. HIV replicationV. HIV treatmentVI. ViroidsVII. Prions

Eukaryotic viruses, viroids, and prions:

257I. Polio virus

A. Basic properties

nonenvelopedinfects nerve cells

+ssRNAicosahedral

258+ RNA (plus strand RNA) means that the RNA genome reads the same as the mRNA

mRNA 5' G G U U C C A A 3'

+ RNA 5' G G U U C C A A 3'

259

nerve cell

1. penetration and uncoating

nucleus of cell

uncoating+ssRNA

B. Life cycle

cytoplasm

2602. Genome replication

+RNA (genome)

-RNA

viral RNA-dependentRNA polymerase

2613. Gene expression

+RNA (mRNA) poliogenome

translation (host machinery)

polyproteinauto-proteolysis and proteolysis

coat proteins, proteases, RNA polymerase etc.

262a. Gene expression facts

Polio mRNA can be translated without a eukaryotic 5' cap (methylguanosine cap).

Polio inactivates translation of host mRNAs by destroying thehost protein that recognizes themethylguanosine cap.

2634. Assembly and release

+ strand RNAs are assembled intocapsids and the host cell is lysed.

264II. Flu virus

A. Basic properties

helical capsidinfects mucus membrane cells of the respiratory tract

-ssRNAsegmented genomeenveloped

265

neuraminidase

segmented genome(-RNA)

B. Structure

hemagglutinin

viral envelope

266

- RNA (minus strand RNA) is complementary to the mRNA

mRNA 5' G G U U C C A A 3'

- RNA 3' C C A A G G U U 5'

267C. Key proteins

1. Hemagglutininmediates fusion of the viral envelope to the host cell membrane

2. NeuraminidaseBreaks down sialic acid and assists in budding

268D. Antigenic shift

This can cause dramatic changesin surface antigens and produce new virulent strains.

Major changes in viral proteins dueto mixing of genome segments from different viruses.Occurs when two different viruses infect the same host.

269III. HIV (AIDS) virusHuman immunodeficiency virus

Causes AIDS

HIV kills CD4+ cells of the immune system

Healthy adults have about 800 CD4+ T-cells/cubic millimeter of blood.

HIV patients are said to have AIDS whenthey develop opportunistic infections or when their CD4+ T-cell count falls below 200.

270A. HIV infection

No cure

Almost always fatal

Usually acquired by sexual intercourse

No vaccine

In the US,~1/250 people are infected.

In the US,~1/3,000 people contract HIV each year

On average, 8-10 years pass between HIV infection and the development of AIDS.

271B. PreventionCelibacyInsistence on condoms Clean needles

4-weeks of treatment with possible side effects of headache, nausea, fatigue and anemia.

Post-exposure drug treatment within 24 h ???

272C. HIV replicationHIV is a retrovirus = an RNA virus that replicates through a DNA intermediate.

reversetranscriptase

HIV genome+ ssRNA(2 copies)

DNA

273D. HIV structure

envelope protein

reverse transcriptase

integrase

protease

+ssRNA

274

gag pol env other genes

LTR = long terminal repeat LTR

Genetic map of typical retrovirus

gag: encodes internal structural proteinspol: encodes reverse transcriptaseenv: encodes envelope proteinsThere are also other genes specific to different retroviruses.

275A. HIV proteins

1. Envelope protein: mediates binding to CD4 receptor

2. Reverse transcriptase: synthesizes DNA from an RNA template

2763. Integrase:

splices viral DNA into the host genome

4. Protease cleaves the viral polyprotein into active parts

277E. HIV reproductive cycle

CD4 receptor

cell membrane

HIV provirus

b

c

e

f

nuclearmembrane

ga

d

278

a. penetration and uncoatingb. reverse transcriptionc. integrationd. gene expression e. replication f. polyprotein cleavage by HIV proteaseg. assembly and budding

Steps in the HIV reproductive cycle

279F. HIV Treatment

http://www.hivatis.org/trtgdlns.html(The latest information on HIV treatment)

In general, two reverse transcriptaseinhibitors are used in combinationwith a protease inhibitor; however,treatment is complex and rapidly changing.

A. Reverse transcriptase inhibitorsB. protease inhibitors

280G. HIV drug resistance

HIV protease

proteaseinhibitor

inhibitor binding to the active siteinactivates the protease

mutation inhibitor nolonger binds butprotease stillfunctions

drugresistantprotease

281VI. Viroids

circular single stranded RNA molecules that cause plant diseases

viroids are "naked" RNA(no proteins associated with RNA)

viroid genomes do NOT encode proteins

282VII. Prions

Infectious proteins

Prion proteins appear to transmit disease without DNA or RNA.

283A. Prion diseases (spongiform encephalopathies)

Scrapie, sheep and goatsMad cow disease, cowsCreutzfeldt-Jacob, humans

284Mad cow disease (BSE)• Bovine spongiform encephalopathy (BSE)• source of infection appears to be feeding

cows with "meat-and-bone meal" remains of infected sheep or cows, especially infected brain tissue

• prion is not destroyed by cooking

285"new variant" Creutzfeldt-Jacob syndrome• human disease thought to be caused by eating BSE-infected beef• about 92 cases, most victims have died• unusual in that many victims are < 30 years old• incubation time is 10 to 15 years

286

normal PrP (prion protein)

disease causing PrP

Disease-causing PrP catalyzesa conformational change that turns normal PrP into disease causing PrP.

Over time, disease causing PrP accumulates and symptoms result.

B. How prions cause disease

287

If a protein transmits the disease, where is its gene?

The prion gene (prp) turned out to be anormal gene found in animals.

Unusual forms of the gene (mutants)are thought to cause disease.

C. The prion gene

288Study objectives1. Describe the structure of the polio virus. Explain polio virus replication and gene expression. What are polyproteins?2. Distinguish between plus strand and minus strand RNA genomes.3. Describe the structure of the flu virus. What is the relationship of flu virus genome structure to antigenic shift. 4. What are the functions of hemagglutinin and neuraminidase.5. How is HIV transmitted? 6. How is HIV infection prevented?7. How is HIV infection treated?8. Describe the structure of HIV. What is a retrovirus? Describe the general structure of a retroviral genome and the proteins encoded.9. Describe the HIV reproductive cycle. Know the functions of the HIV proteins.10. What are viroids? How do viroids differ from viruses and prions?11. What are prions?12. What diseases do prions cause?13. How are prions thought to cause disease? 14. Where are prions genes found?

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MCB 3020, Spring 2005 Chapter 7: Molecular Genetics