gene expression at the overview of gene expression...

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Chapter 12Gene Expression at the

Molecular Level

n Overview of Gene Expressionn Transcriptionn RNA Processing in Eukaryotesn Translation and the Genetic Coden The Machinery of Translationn The Stages of Translation

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Key Concepts:n We can look at gene function at two levels:

q molecular function of the protein productq organism’s trait conferred by the gene

n Two levels are connected – the molecular function affects the structure and function of cells to determine the trait

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Overview of Gene Expression

n 1908, Archbold Garrod first proposed relationship between genes and the production of enzymes

n Studied patients with metabolic defects, or “inborn errors of metabolism”

n Alkaptonuriaq Patient’s body accumulates abnormal levels of homogentisic

acid (alkapton)q Recessive pattern of inheritance

n Hypothesis: “Disease is due to missing enzyme”n The nature of the genetic material was completely

unknown at the time3

Inborn errors of metabolism

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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

NH2

C

H

C

H

Tyrosineaminotransferase

Phenylalaninehydroxylase

Hydroxyphenylpyruvateoxidase

Homogentisicacid oxidase

Maleylacetoaceticacid

Alkaptonuria

Tyrosinosis

Homogentisicacid

p-Hydroxyphenylpyruvicacid

Tyrosine

COOHHO CH2

NH2

Phenylalanine

COOH

Enzyme

CH2

Disorder

Phenylketonuria

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n Early 1940s – Beadle and Tatum became aware of Garrod’s work

n They worked on Neurospora crassa, common bread mold

n Minimum requirements for growthq Carbon source (sugar), inorganic salts, and biotinq Neurospora can synthesize everything else it needs

from those precursors

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Beadle and Tatumn Wildtype – normal Neurospora, can grow on

minimal medium

n Mutant strains – unable to grow unless supplemented with specific substances (vitamins or amino acids)

n Each single mutation resulted in the requirement for a single type of vitamin

n Hypothesis: “One gene, one enzyme”

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n Multiple mutants required arginine to grown Could they grow if supplemented with precursors instead?n Fell into three groups based on requirements

n Conclusion – Supports one gene, one enzyme hypothesis7

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1

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1

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Citrulline ArgininePrecursormolecule Enzyme 1 Enzyme 2 Enzyme 3

Ornithine

(a) Simplified pathway for arginine synthesis

(b) Growth of strains on minimal and supplemented growth media

WT

Neurosporagrowth

WT WT WT

Minimal +Ornithine +Citrulline +Arginine


n A modification of the “one gene, one enzyme”hypothesis

n Enzymes are only one category of cellular proteins – genes also encode other proteins

n Also, some proteins are composed of several polypeptides that work together for one function

nex: Hemoglobin composed of 4 polypeptides

n “One gene, one polypeptide”

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Modern understanding

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Central dogma

n Transcriptionq Produces a transcript (RNA copy) of a geneq This messenger RNA (mRNA) specifies the amino

acid sequence of a polypeptide

n Translationq Process of synthesizing specific polypeptide on a

ribosome using the mRNA template

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DNA RNA Protein

n Eukaryotes also have an intervening step called RNA processing where pre-mRNA is processed into active mRNA

n Some genes do not encode polypeptides – an RNA is the final functional productq Structural RNAsq Regulatory RNAs

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DNA pre-mRNA mRNA Protein

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Nucleus

Polypeptide

CytoplasmCytosol

PolypeptideRibosome

Ribosome

(b) Molecular gene expression in eukaryotes

(a) Molecular gene expression in prokaryotes

Transcription:DNA is transcribedinto an RNA copy.

Translation:mRNA is translatedInto a polypeptideat the ribosome.

mRNA

DNA

DNA

Pre-mRNAmRNA

Translation

Transcription

RNA processing

n Genes constitute the genetic materialq The “blueprint” for organisms’ characteristics

n Structural genes code for polypeptides

n One or several polypeptides act as a protein to play some role in the cell

n Activities of proteins determine the structure and function of cells

n Cellular activity, taken together in an organism determines their traits or characteristics

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How do genes determine traits?

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n Gene:q An organized unit of DNA sequences that enables

a segment of DNA to be transcribed into RNA and ultimately results in the formation of a functional product

q Other genes code for RNA itself as a productn Transfer RNA (tRNA) - translates mRNA into amino acidsn Ribosomal RNA (rRNA) - part of ribosomes

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Transcription

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Transcribed region: Part of this regioncontains the information that specifiesan amino acid sequence.

Transcription

mRNA

DNA

3′5′

Promoter:Signals thebeginning oftranscription.

Terminator:Signals the endof transcription.

Regulatory sequence: Sitefor the binding of regulatoryproteins. The role of regulatoryproteins is to influence the rateof transcription.


Three stages of transcription

1. Initiationq Recognition stepq In bacteria, sigma factor causes RNA polymerase

to recognize promoter regionq Stage completed when DNA strands separate

near promoter to form open complex

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ElongationInitiation Termination 2. Elongationq RNA polymerase synthesizes RNAq Template or coding strand used for RNA synthesis

n Noncoding strand is not used

q Synthesized 5’ to 3’q Uracil substituted for thymine

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3. Terminationq RNA polymerase reaches termination sequenceq Causes both the polymerase and newly-made RNA

transcript to dissociate from DNA

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5′ end

Rewinding oftranscribed DNA(upstream)

RNApolymerase

Promoter

5′

3′

Sigmafactor

Opencomplex

Directionof travel

Unwinding ofentering DNA(downstream)

3¢ end(growing end)

Terminator

CompletedmRNA transcript

A T C G GU A G C C

TemplateDNA strand

5′Directions ofRNA synthesis

3′ 3′

Initiation:The promoter functions as a recognition sitefor sigma factor. RNA polymerase is bound tosigma factor, which causes it to bind to thepromoter. Following binding, the DNA isunwound to form an open complex.

1 Elongation/synthesis of the RNAtranscript:Sigma factor is released, and RNApolymerase slides along the DNA inan open complex to synthesize RNA.

2 Termination:When RNA polymerase reachesthe terminator, it and the RNAtranscript dissociate from theDNA.

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mRNAtranscript

n Direction of transcription and which DNA strand used varies among genes

n In all cases, synthesis of RNA transcript is 5’ to 3’and DNA template strand reads 3’ to 5’

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5¢3¢

3¢

5¢ 3¢

5¢5¢3¢

3¢

5¢ 3¢

5¢5¢3¢

3¢

3¢ 5¢

5¢

Template strand

Chromosomal DNA

Promoter TerminatorPromoterTerminator

Gene A Gene B Gene C

Gene A RNA Gene B RNA Gene C RNA

TerminatorPromoter

Template strandDirection oftranscription

Regions of DNAbetween genes

Eukaryotic transcription

n Basic features identical to prokaryotes

n However, each step has more proteins

n Three forms of RNA polymeraseq RNA polymerase II – transcribes mRNAq RNA polymerase I and III – transcribes nonstructural

genes for rRNA and tRNA

n RNA polymerase II requires 5 general transcription factors to initiate transcription

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n Bacterial mRNAs can be translated immediately

n Eukaryotic mRNAs are made in a longer pre-mRNA form that requires processing into mature mRNAq Introns – transcribed but not translatedq Exons – coding sequence found in mature mRNA

n Splicing – removal of introns

n Other modifications – addition of tails and caps

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RNA Processing in Eukaryotes

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Promoter

Exon 1

Exon 1

Exon 2

Exon 2

Exon 3

Exon 3

Eukaryotic structural gene

Intron 1 Intron 2

5¢

5¢ cap

3¢Capping

Splicing

5¢ 3¢

Introns removed

TerminatorTranscription

Tailing

3¢ poly A tail

Pre-mRNA

Mature mRNA

Splicing

n Introns found in many eukaryotic genesq Most structural genes have one or more introns

n Spliceosome – removes introns preciselyq Composed of snRNPs (small nuclear RNA + proteins)

n Alternative splicing – splicing can occur more than one way to produce different products

n rRNA and tRNA are self-splicingq They are ribozymes – RNAs that can catalyze

reactions23 24


The first 2 snRNPsubunits bind to the5′ splice site andbranch site.

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Additional snRNPsubunits bind to the 3′splice site and otherlocations to create a loop.

2

3′5′

3′

3′

5′

3′5′

3′5′

5′

The 5′ splice site is cut.The 5′ end of intron iscovalently attached tothe branch site. TwosnRNP subunits arereleased.

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The 3′ splice site iscut. Exon 1 is covalentlyattached to exon 2. Theintron (in the form of aloop) is released alongwith the rest of the snRNPsubunits and degraded.

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5′ splicesite Exon

Intron

Branch site 3′ splicesite

Exon

Spliceosome

Branchsite

Intron plussubunits

Exon 1

Mature mRNAExon 2

Pre-mRNA

snRNPsubunits

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Additional RNA processing

n Cappingq Modified guanosine attached to 5’ endq Needed for mRNA to exit nucleus and bind ribosome

n Poly A tailq 100-200 adenine nucleotides added to 3’ endq Increases stability and lifespan in cytosolq Not encoded in gene sequence

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N+

O P CH2

O–

O

HH

OH

H

O

O

P CH2

O–

Base

H

O

O

O

O

P

O–

OO

OHOOHP

O–

CH2

CH3

HH

H H

H H

N

N

3¢

5¢

3¢

5¢

5¢

3¢

N

O

O

H2N

7-methylguanosine cap

(a) Cap structure at the 5¢ end of eukaryotic mRNA

Poly A tail(b) Addition of a poly A tail at the 3¢ end of eukaryotic mRNA

A poly A tail consisting of 100–200adenine nucleotides is added aftertranscription.

A

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n Genetic code – sequence of bases in an mRNA molecule

n Read in groups of three nucleotide bases or codons

n Most codons specify a particular amino acidq Also Start and Stop codons

n Degenerate code – more than one codon can specify the same amino acid

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Translation and the Genetic Code

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n Bacterial mRNA has 5’ ribosomal-binding siten Start codon usually AUGn Typical polypeptide is a few hundred amino acids in lengthn Stop codons (aka Termination or nonsense codons)

q UAA, UAG or UGA 30

Ribosomal-binding site:The site for ribosomebinding.

Start codon: A codon thatspecifies the first amino acidin a polypeptide sequence.

5′ 3′

Stop codon: Specifiesthe end of translation.

Coding sequence: A series of codonsfrom the start codon to the stop codonthat determine the sequence of aminoacids of a polypeptide.

Polypeptide

Translation

mRNA


Reading frame

n Start codon defines reading frame

n Addition of a U shifts the reading frame and changes the codons and amino acids specified

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5’ –AUAAGGAGGUUACG(AUG)(CAG)(CAG)(GGC)(UUU)(ACC) – 3’Met –Gln -Gln -Gly -Phe -Thr

5’ –AUAAGGAGGUUACG(AUG)(UCA)(GCA)(GGG)(CUU)(UAC)C –3’

Met –Ser -Ala -Gly -Leu -Tyr

n mRNA q Codon – set of 3 RNA nucleotidesq T of DNA substituted for U of RNA

n tRNA q Anticodon – 3 RNA nucleotide part of tRNA moleculeq Allows binding of tRNA to mRNA codon

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Coding strand

Polypeptide

Codon

Anticodon

5′

3′

5′

3′

5′

5′

3′

A C T G C C C A A A AT G A G C G G G G G G G G GA C C C C C C CT T T T

A C U G C C C A A A AU G A G C G G G G G G G G GA C C C C C C CU U U U A GCU

T G A C G G G T T T TA C T C G C C C C C C C C CT G G G G G G GA A A AA GCTT CGA

U UA C U C C C C C C C C CU G G GA A A

Met Ser Asp Pro Phe Gly Leu Gly Glu

G G G G G U

COO–NN3+

Transcription

Translation

DNA

mRNA

N-terminus(amino terminus)

Untranslatedregion

Startcodon

3′ Untranslatedregion

tRNA

C-terminus(carboxyl terminus)

Peptidebond

Aminoacids

Template strand

Stopcodon

Nirenberg and Leder Found the RNA Triplets Can Promote the Binding of tRNA to Ribosomes

n 1964 – found that an RNA triplet can act like a codon within an mRNA molecule

n Experiment establishes relationship between triplet sequence and specific amino acids

n Used radiolabeled amino acids bound to tRNA

n Complex of tRNA, RNA triplet and ribosome could be filtered by size


Experimental level Conceptual level

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2

3

HYPOTHESIS

Ribosome

Filter

Radiolabeled proline

Filter

Proline

An RNA triplet can bind to a ribosome and promote the binding of the tRNA that carries the amino acidthat the RNA triplet specifies.

The researchers made 20 in vitro translation systems, which included ribosomes, tRNAs, and 20 amino acids. The 20 translation systems differed with regard to which amino acid was radiolabeled. For example, in 1translation system, radiolabeled glycine was added, and the other 19 amino acids were unlabeled. In anothersystem, radiolabeled proline was added, and the other 19 amino acids were unlabeled. The in vitro translationsystems also contained the enzymes that attach amino acids to tRNAs.

Mix together RNA triplets of a specificsequence and 20 in vitro translationsystems. In the example shown here, the triplet is 5¢–CCC– 3′. Eachtranslation system contained a differentradiolabeled amino acid. (Note: Only 3tubes are shown here.)

Allow time for the RNA triplet to bindto the ribosome and for the appropriate tRNA to bind to the RNA triplet.

Pour each mixture through a filter thatallows the passage of unbound tRNAbut does not allow the passage ofribosomes. Ribosomes

trappedon filter

In vitro translationsystem with 1radiolabeled amino acid (for example, proline)

Tubes containingan RNA triplet

Proline tRNA

RNA triplet thatspecifies proline

KEY MATERIALS

tRNAs not bound to a ribosome


6 CONCLUSION This method enabled the researchers to identify many of the codons of the genetic code.

7 Leder, Philip, and Nirenberg, Marshall W . 1964. RNA Codewords and Protein Synthesis, III. On thenucleotide sequence of a cysteine and a leucine RNA codeword. Proceedings of the National Academy of Science 52:1521-1529.

SOURCE

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5

Scintillation counter

5′ – AAA – 3′

5′ – ACA – 3′

5′ – ACC – 3′

5′ – AGA – 3′

5′ – AUA – 3′

5′ – AUU – 3′

5′ – CCC – 3′

5′ – CGC – 3′

5′ – GAA – 3′

Threonine

Threonine

Arginine

Isoleucine

Isoleucine

Proline

Arginine

Glutamic acid

Aspartic acid

Alanine

Glycine

Glycine

Cysteine

Leucine

Count radioactivity on the filter.

THE DATA

TripletRadiolabeled amino acidtrapped on the filter

Lysine

Valine

Tyrosine

Radiolabeled amino acidtrapped on the filterTriplet

5′ – UUG – 3′5′ – UGU – 3′

5′ – UAU – 3′5′ – GUU – 3′

5′ – GGC – 3′5′ – GGU – 3′

5′ – GCC – 3′5′ – GAC – 3′

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n Requires many componentsq mRNAq tRNAq ribosomesq translation factors

n Most cells use a substantial amount of energy on translation

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The Machinery of Translation tRNA

n Different tRNA molecules encoded by different genes

n tRNASer carries serine

n Common featuresq Cloverleaf structureq Anticodonq Acceptor stem for

amino acid binding

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Amino acid attachmentsite at the 3′ single-strandedregion

Stem-loopstructure

Anticodon

(a) Two-dimensional structure of tRNA

3′

5′

G G C

3′ singlestranded

region

(b) Three-dimensional structure of tRNA

Anticodon

5′

Hydrogen bonds

Aminoacyl-tRNA synthetase

n Catalyzes attachment of amino acids to tRNAq One for each of 20 different amino acids

n Reactions result in tRNA with amino acid attached (charged tRNA or aminoacyl tRNA)

n Ability of aminoacyl-tRNA synthetase to recognize appropriate tRNA has been called the “second genetic code”

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A specific amino acidand ATP bind toaminoacyl-tRNAsynthetase.

Amino acidATP

tRNA

Pi

P

PP

PyrophosphateAMP

PPPCharged

tRNA

Aminoacyl-tRNAsynthetase

1 The amino acid is activatedby the covalent binding ofAMP, and pyrophosphateis released.

2 The correct tRNAbinds to the synthetase.The amino acid iscovalently attached tothe tRNA. AMP isreleased.

3 The chargedtRNA is released.

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Ribosomes

n Prokaryotes have one kind of ribosome

n Eukaryotes have distinct ribosomes in different cellular compartmentsq Here, we focus on cytosolic ribosomes

n Composed of large and small subunits

n Structural differences between prokaryotes and eukaryotes exploited by antibiotics to inhibit bacterial ribosomes only

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n Overall ribosome shape determined by rRNA

n Discrete sites for tRNA binding and polypeptide synthesisq P site – Peptidyl siteq A site – Aminoacyl siteq E site – Exit site

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3′5′

E P A

(b) Schematic model for ribosome structure(a) Bacterial ribosome model based on X-ray diffraction studies

50S subunit

E site

P site

30S subunit

Polypeptide

E site

P site

30S subunit

50S subunit

tRNA

A site

mRNA

A site


a: Reprinted from Seth A. Darst, “Bacterial RNA polymerase,” Current Opinion in Structural Biology, 11(2):155–62, © 2001, with permission from Elsevier

1. Initiationq mRNA, first tRNA and ribosomal subunits assemble

2. Elongationq Synthesis from start codon to stop codon

3. Termination q Complex disassembles at stop codon releasing the

completed polypeptide

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The Stages of Translation

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5′

3′

Initiation: mRNA, tRNA, andthe ribosomal subunits forma complex.

Elongation: The ribosometravels in the 5′ to 3′direction and synthesizesa polypeptide.

Termination: The ribosomereaches a stop codon, andall of the componentsdisassemble, releasing acompleted polypeptide.

1 2 3

Initiator tRNA Polypeptide

Start codon

Small subunit

mRNA

Large subunit

Stop codon

Completedpolypeptide

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Initiation

n mRNA, the first tRNA and ribosomal subunits assemble

n Requires help of ribosomal initiation factors

n Also requires input of energy (GTP hydrolysis)

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n Initiation in bacteriaq mRNA binds to small ribosomal subunit facilitated

by ribosomal-binding sequenceq Start codon is a few nucleotides downstreamq Initiator tRNA recognizes start codon in mRNAq Large ribosomal subunit associatesq At the end, the initiator tRNA is in the P site

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Initiation


5¢3¢

5¢3¢

Start codon (AUG)

E P A

G

GCAU

mRNA

Ribosomal-bindingsite

Small (30S)ribosomalsubunit

mRNA-bindingsequence of16S rRNA

Initiator tRNA

Large (50S)ribosomalsubunit

70S initiationcomplex

mRNA binds to thesmall ribosomalsubunit.

1

Initiator tRNAbinds to the startcodon in mRNA.

2

Large ribosomalsubunit binds.

3

GCAU

5¢ 3¢

n Two eukaryotic differences in initiationq Instead of a ribosomal-binding sequence, mRNAs

have guanosine cap at 5’ endn Recognized by cap-binding proteins

q Position of start codon more variablen In many cases, first AUG codon used as start codon

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Elongation

1. Aminoacyl tRNA brings a new amino acid to the A site

q Binding occurs due to codon / anticodon recognition

q Elongation factors hydrolzye GTP to provide energy to bind tRNA to A site

q Peptidyl tRNA is in the P siteq Aminoacyl tRNA is in the A site

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2. Peptide bond is formed between the amino acid at the A site and the growing polypeptide chain

q The polypeptide is removed from the tRNA in the P site and transferred to the amino acid at theA site

q Called the peptidyl transfer reactionq rRNA catalyzes peptide bond formation (the

ribosome is a ribozyme)

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3. Movement or translocation of the ribosome toward the 3’ end of the mRNA by one codon

q Shifts tRNAs at the P and A sites to the E and P sites

q The next codon is now at the A spotq Uncharged tRNA exits from E spot

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Elongation


tRNA entry: Acharged tRNAbinds to the A site.

1

Peptidyl transferreaction: A bondforms between thepolypeptide chainand the aminoacid in the A site.The polypeptide istransferred to theA site.

2

Translocation ofribosome andrelease of tRNA:The ribosometranslocates 1codon to the right.The unchargedtRNA is releasedfrom the E site.This process isrepeated again andagain until a stopcodon is reached.

3

Anticodon

50S subunit

30S subunit

E P A

ChargedtRNA

Polypeptide emerging from exit channel

mRNA

5¢ 3¢

5¢ 3¢

5¢ 3¢

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Termination

n When a stop codon is found in the A site, translation ends

n 3 stop codons – UAA, UAG, UGAn Recognized by release factors

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n Completed polypeptide is attached to a tRNA in the P site and stop codon in the A site

1. Release factor binds to stop codon at the A site2. Bond between polypeptide and tRNA hydrolyzed to

release polypeptide3. Ribosomal subunits and release factors disassociate

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Termination


Polypeptide

Stop codon5¢ 3¢

E P AReleasefactor

5¢ 3¢

5¢

3¢+

50Ssubunit

30Ssubunit

mRNA

The mRNA, ribosomalsubunits, and releasefactor dissociate.

A release factorbinds to the stopcodon at the Asite.

The polypeptideis released fromthe tRNA in theP site. The tRNAis then released.

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2

3

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Structure of Klenow Taq1

gene expression at the overview of gene expression...

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