Chapter 17: From Gene to Protein

• Gene: A segment of DNA that specifies the amino acid sequence of a polypeptide

• DNA does not directly control protein synthesis, instead its information is transcribed into RNA

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Overview: The Flow of Genetic Information

• The information content of DNA– Is in the form of specific sequences of

nucleotides along the DNA strands– http://www.dnai.org/text/mediashowcase/index2.html?id=588

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Genes specify proteins via transcription and translation

• The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins

• The process by which DNA directs protein synthesis is called gene expression– Includes two stages, called transcription and translation

• The Central Dogma of Molecular Genetics:– There are 3 major classes of genetic biopolymers: DNA and RNA

(both nucleic acids), and protein.

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One Gene, One Enzyme hypothesis• Synthesis of all substances in living things is dictated by enzymes

– Remember that enzymes are proteins whose 1' structure (sequence of linked amino acids) are coded for by DNA base triplets.

• Beadle and Tatum experiments (1941)

• Purpose: "to determine if and how genes control known biochemical reaction" – Work with red bread mold Neurospora crassa to find “nutritional

mutants”– Used radiation to create “auxotrophs”, organisms, such as a strain

of bacteria, that have lost the ability to synthesize certain substances required for its growth and metabolism as the result of mutational changes.

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Beadle and Tatum’s experiment

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X-ray mutations loss of enzyme lack of an AA (ex. Arg.) mold could only grow on arginine-supplemented media

Beadle and Tatum proposed that a single gene (thru a single mutation) codes for a single specific enzyme = Nobel Prize (1958)

The Products of Gene Expression: A Developing Story

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• Beadle and Tatum developed the :"One Gene - One Enzyme"  correlation:– Which states that the

function of a gene is to dictate the production of a specific enzyme

– Later found out not necessarily true: only some proteins are enzymes.   It is also true of structural proteins, chains of polypeptides, or hormones.

Ribonucleic Acid• Why would the cell want to have an intermediate

between DNA and the proteins it encodes? – The DNA can then stay pristine and protected,

away from the caustic chemistry of the cytoplasm.

– Gene information can be amplified by having many copies of an RNA made from one copy of DNA.

– Regulation of gene expression can be effected by having specific controls at each element of the pathway between DNA and proteins.

– The more elements there are in the pathway, the more opportunities there are to control it in different circumstances. 7

24.2 Gene Expression

• RNA (ribonucleic acid)

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24.2 Gene Expression

• Three Classes of RNA– Messenger RNA (mRNA)

• Takes a message from DNA to the ribosomes• strand

– Ribosomal RNA (rRNA)• Makes up ribosomes (along with proteins)• globular

– Transfer RNA (tRNA)• Transfers amino acids to ribosomes• Hairpin shape

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24.2 Gene Expression

• Gene Expression Requires Two Steps:• Transcription

– Is the synthesis of RNA under the direction of DNA– Produces messenger RNA (mRNA)

• Translation– Is the actual synthesis of a polypeptide, which occurs

under the direction of mRNA– Occurs on ribosomes

http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation1.html

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24.2 Gene Expression

• Transcription– During transcription, a segment of the DNA serves as a template

for the production of an RNA molecule

– Messenger RNA (mRNA)• RNA polymerase (enzyme) binds to a promoter (“start”

sequence)• DNA helix is opened so complementary base pairing can

occur• RNA polymerase joins new RNA nucleotides in a sequence

complementary to that on the DNA, in a 5’ to 3’ direction

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Transcription of DNA to form mRNA

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Messenger RNA

• mRNA - of the 64 possible 3-base combinations:– 61 code for the twenty different amino acids – 3 code for "stop"; i.e. chain termination

• Specific nucleotide sequences call for “start” of transcription (usually  AUG = methionine) = PROMOTOR sequence

• “stop” of mRNA synthesis = TERMINATION sequence (UAA, UGA, UAG)

• Finished mRNA strands are ~500-10,000 nucleotides long

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Cracking the Code• A codon in messenger RNA

– Is either translated into an amino acid or serves as a translational stop signal

Figure 17.5

Second mRNA baseU C A G

U

C

A

G

UUUUUCUUAUUG

CUUCUCCUACUG

AUUAUCAUAAUG

GUUGUCGUAGUG

Met orstart

Phe

Leu

Leu

lle

Val

UCUUCCUCAUCG

CCUCCCCCACCG

ACUACCACAACG

GCUGCCGCAGCG

Ser

Pro

Thr

Ala

UAUUAC

UGUUGC

Tyr Cys

CAUCACCAACAG

CGUCGCCGACGG

AAUAACAAAAAG

AGUAGCAGAAGG

GAUGACGAAGAG

GGUGGCGGAGGG

UGGUAAUAG Stop

Stop UGA StopTrp

His

Gln

Asn

Lys

Asp

Arg

Ser

Arg

Gly

U

CA

GUCAG

UCAG

UCAG

Fir

st m

RN

A b

ase

(5

en

d)

Th

ird

mR

NA

bas

e (3

e

nd

)

Glu14

• During transcription– The gene determines the sequence of bases along

the length of an mRNA molecule

Figure 17.4

DNAmolecule

Gene 1

Gene 2

Gene 3

DNA strand(template)

TRANSCRIPTION

mRNA

Protein

TRANSLATION

Amino acid

A C C A A A C C G A G T

U G G U U U G G C U C A

Trp Phe Gly Ser

Codon

3 5

35

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The Process of Transcriptionhttp://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation20.html

transfer RNASmall, ~80 nucleotides long.  tRNA exists as a

single-stranded molecule. However, regions of double helix can form where there is some base pair complementation (U and A , G and C), resulting in hairpin loops. The RNA molecule with its hairpin loops is said to have a secondary structure.

It can bind an amino acid at one end, and mRNA (anticodon) at the other end. It acts as an adaptor to carry the amino acid elements of a protein to the appropriate place as coded for by the mRNA codon (complementary).

The "Wobble Phenomenon": There are only 40 different types of t-RNA and 64 codons. This means that some of the t-RNA can pair up with several different codons. This can occur because there is some third base “flexibility”.

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Transfer RNA: Amino Acid Carrier

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rRNA

– Ribosomal RNA is the most abundant type of RNA in cells

– Ribosomes: comprised of subunits 2/3 RNA, 1/3 protein

• Two populations of ribosomes are evident in cells, Free and bound• Free ribosomes in the cytosol initiate the synthesis of all proteins

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• The ribosome has three binding sites for tRNA– The P site– The A site– The E site

Figure 17.16b

E P A

P site (Peptidyl-tRNAbinding site)

E site (Exit site)

mRNAbinding site

A site (Aminoacyl-tRNA binding site)

Largesubunit

Smallsubunit

Schematic model showing binding sites. A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites. This schematic ribosome will appear in later diagrams.

(b)

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Translation (Building a polypeptide) requires Three Steps:

– Initiation (requires energy)– Elongation (requires energy)– Termination

Amino end Growing polypeptide

Next amino acidto be added topolypeptide chain

tRNA

mRNA

Codons

3

5

Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site.

(c)

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Animation: How Translation Works.http://highered.mcgraw-hill.com/sites/0072507470/student_view0/chapter3/animation__how_translation_works.html

Proteins: A review of structure

(alpha) helix

C NCH

R

C

CH

R

C N

C

CH

R

C

N

C

CH

R

N

C

CH

R

NCH

R

N

C

NCH

R

CH

a. primary structure

b. secondary structure

c. tertiary structure

H3N+

COO–

CO

N HCR

HCN

C

C RCO

R CN H

C ONH

ONC

H O CC

N H

OC

CR

N

N

R

H

C

C

ON

CCR

C

H

O

HOCNCC

N HRC

C O

NHCC

R

HRO

(beta) sheet = pleated sheet

O

O

O

O

O

OHO

H

C

H

H

H

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

• Concept 17.4: Translation is the RNA-directed synthesis of a polypeptide: a closer look

Narrated animation: Protein Synthesis (with quiz) http://highered.mcgraw-hill.com/sites/0072507470/student_view0/chapter3/animation__protein_synthesis__quiz_3_.html

Interactive practice: Transcribe & Translate a Gene http://learn.genetics.utah.edu/content/begin/dna/transcribe/

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Polyribosomes– Several ribosomes may move along the same mRNA

• Multiple copies of a polypeptide may be made• The entire complex is called a polyribosome

Figure 17.20a, b

Growingpolypeptides

Completedpolypeptide

Incomingribosomalsubunits

Start of mRNA(5 end)

End of mRNA(3 end)

Polyribosome

An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes.

(a)

Ribosomes

mRNA

This micrograph shows a large polyribosome in a prokaryotic cell (TEM).

0.1 µm(b)

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Overview of Gene ExpressionSimple Gene Expression animationhttp://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter15/simple_gene_expression.html

Detailed Protein Synthesis animationhttp://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/micro06.swf::Protein%20Synthesis

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Summary of Gene Expression

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Regulation of gene expression

• Genes are activated in some cells, but not others• Genes can be active some of the time, but not others

• The mechanics of the “on/off” switch for genes was first identified in bacteria.

• 1965 Nobel Prize in Medicine François Jacob, Jacques Monod and André Lwoff.

• This operon enables the metabolism of lactose in Escherichia coli

Animation of the lac operon http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter15/the_lac_operon.html

Regulation of Gene Expression in Eukaryotes

4 levels:1. Transcriptional control (nucleus):

• e.g. chromatin density and transcription factors

2. Posttranscriptional control (nucleus)• e.g. mRNA processing

3. Translational control (cytoplasm)• e.g. differential ability of mRNA to bind ribosomes

4. Posttranslational control (cytoplasm)• e.g. changes to the protein to make it functional

Animation: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120080/bio31.swf::Control%20of%20Gene%20Expression%20in%20Eukaryotes

Regulation of gene expressionTranscriptional control (nucleus):

– e.g. chromatin density and transcription factors

– Euchromatin: Loosely packed form of DNA; genes are transcibed– Heterochromatin: tightly packed form of DNA; genes are “silenced”

– A transcription factor (sometimes called a sequence-specific DNA-binding factor) is a protein that binds to specific DNA sequences, thereby controlling the flow (or transcription) of genetic information from DNA to mRNA. Transcription factors perform this function alone or with other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase (the enzyme that performs the transcription of genetic information from DNA to RNA) to specific genes.

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Transcription factors in Prokaryotes

InductionAnimation: The lac operon inductionhttp://highered.mcgraw-hill.com/sites/0073377988/student_view0/chapter21/the_lac_operon__induction_.html

RepressionAnimation: The trp operonhttp://highered.mcgraw-hill.com/sites/0072995246/student_view0/chapter7/the_trp_operon.html

Transcription factors in Eukaryotes, animation/quiz http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter15/transcription_factors.html

Posttranscriptional control (nucleus) Processing of mRNA

• After Transcription

• Primary “Pre-”mRNA must be modified into mature

mRNA– Introns are intragene segments (often, junk)

– Exons are the portion of a gene that is expressed

• Intron sequences are removed, and a poly-A tail is

added– Ribozyme splices exon segments together

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Eukaryotic mRNA modification: RNA splicing animationhttp://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation22.html

mRNA Processing pre-RNA must be modified before translation

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The Functional and Evolutionary Importance of Introns

• The presence of introns– Allows for alternative RNA splicing– Additional animations of RNA processing:

• Processing of Gene Information: Prokaryotes –vs- Eukaryotes: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/bio25.swf::Processing%20of%20Gene%20Information%20-%20Prokaryotes%20versus%20Eukaryotes

• How Spliceosomes Process RNA: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/bio30.swf::How%20Spliceosomes%20Process%20RNA

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Transposons

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• “jumping genes”• Sections of DNA that can move to new locations and disrupt gene sequences• Animation http://highered.mcgraw-hill.com/sites/0073377988/student_view0/chapter21/transposons__shifting_segments_of_the_genome.html

See Barbara McClintock

Chromosome 11 flyover