Chapter 17

Gene Expression: From Gene to Protein

17.1 Genes specify proteins via transcription and translation

The concept of the gene has changed over time

• Studies led to a one gene-one protein hypothesis

• It became clear that a mutant phenotype arises from a change in the protein’s amino acid sequence because proteins are polymers of amino acids

• Those ideas were to simple

• Later research refined the one gene - one protein hypothesis.

• Later research demonstrated that many proteins are composed of several polypeptides, each of which has its own gene.

• Therefore, Beadle and Tatum’s idea has been restated as the one gene - one polypeptide hypothesis.

Example of the one gene-one polypeptide hypothesis:

• Hemoglobin contains two types of polypeptide chains, alpha and beta.

• Only the beta chain is affected in persons with sickle-cell trait and sickle-cell disease; therefore there must be a gene for each type of chain.

• Sickle-cell disease results when the 6th of 146 amino acids in the beta chain is changed from glutamate to valine. This causes the Hbs to be less soluble and to precipitate out of solution, distorting the RBCs into the sickle shape.

Genes are expressed via transcription translation

• Genes are expressed as RNAs, some of which are translated into proteins (2 steps)

• Transcription-the information in a DNA sequence is copied into a complementary RNA sequence

• Translation-this RNA sequence is used to create the amino acid sequence of a polypeptide

Good Transcription Translation video

Three types of RNA

• 1. Messenger RNA (mRNA)– A modified RNA strand complementary of a

transcribed DNA strand

– Travels from the nucleus to the cytoplasm, where it is translated into a polypeptide

– The sequence of mRNA determines the amino acid sequence of the polypeptide chain

mRNA

http://crescentok.com/staff/jaskew/isr/anatomy/anatomy1/unit4.htm

• 2. ribosomal RNA (rRNA)– the ribosome is a protein synthesis factory

with multiple proteins and several ribosomal RNAs (rRNAs)

– These form the peptide bond between the amino acids

• 3. transfer RNA (tRNA)– Binds a specific amino acid and recognizes a

specific sequence of nucleotides in mRNA

– It recognizes which amino acid should be added next to a growing polypeptide chain

tRNA

http://www.wiley.com/college/boyer/0470003790/structure/tRNA/trna_intro.htm

17.2 Transcription is the DNA-directed synthesis of RNA: a closer

look• Transcription requires several components:

– A DNA template for complementary base pairing

– The appropriate energy molecules (ATP etc…)

– AN RNA polymerase enzyme

• Transcription produces all the different types of RNA

RNA polymerases share common features

• RNA polymerase-catalyze the synthesis of RNA from the DNA template

• Unlike DNA polymerases, RNA polymerase doesn’t require a primer

RNA polymerase

http://www.rcsb.org/pdb/101/motm.do?momID=40

Transcription occurs in three steps

• 1. Initiation

• 2. Elongation

• 3. Termination

http://www.nature.com/scitable/content/dna-is-transcribed-to-form-rna-44350

Initiation

• Begins with a promoter-a special DNA sequence to which the RNA polymerase binds to

• Promoters tell the RNA polymerase:– Where to start transcription (capitalization at

the beginning of a sentence)

– Which of the two DNA strands to transcribe

Elongation

• RNA polymerase unwinds the DNA and reads the template strand in the 3’-5’ direction

• This creates the new complementary RNA strand in a 5’-3’ direction

• Adds new RNA nucleotides by complementary base pairing based off of the DNA template strand

• Similar to DNA replication except Thymine is replaced with Uracil

• As transcription goes on, the two DNA strands rewind and the RNA grows as a single strand

• Transcription can also make mistakes, but they have proofreading proteins as well

• Not as dangerous as a mistake in DNA

Termination

• Certain base sequences specify termination

• Some genes the newly formed transcript falls away from the DNA template

• Other genes proteins pull the transcript away

Transcription animated Tutorial

• http://www.biotechnologyonline.gov.au/popups/int_transcription.html

17.3 Eukaryotic cells modify RNA after transcription

Eukaryotic Coding Regions

• Coding regions in DNA are expressed as proteins

• In eukaryotes each gene has its own promoter, but not in prokaryotes (usually 1)

• A eukaryotic gene contains noncoding sequences called introns

• The transcribed regions are called exons

• Both introns and exons appear in the primary mRNA transcripts called pre-mRNA

• Introns get removed before the mature mRNA leaves the nucleus

• This is pre-mRNA processing

Eukaryotic gene transcripts are processed before translation

• Primary transcript of a gene is modified before it leaves the nucleus

– Introns are removed

– Both ends of the pre-mRNA are chemically modified

Splicing to Remove Introns

• A Nonfunctional protein would be produced if introns were not cut out

• A process called RNA splicing removes the introns and splices the exons together

• Located between the introns and exons are consensus sequences

• These sequences are bound by small nuclear ribonucleoprotein particles (snRNPs)

• These bind at the ends and pull the two ends together which forms a large RNA-protein complex called a spliceosome

• This cuts out the introns and joins the ends of the exons together

Ribozymes: RNA molecules that function as enzymes, catalyzing their own excision

The Functional and Evolutionary Importance of Introns

• Some introns contain sequences that may regulate gene expression

• Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during splicing

• This is called alternative RNA splicing• Consequently, the number of different proteins

an organism can produce is much greater than its number of genes

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• Proteins often have a modular architecture consisting of discrete regions called domains

• In many cases, different exons code for the different domains in a protein

• Exon shuffling may result in the evolution of new proteins

© 2017 Pearson Education, Inc.

GeneDNA

Exon 1

Transcription

RNA processing

Translation

Domain 3

Domain 2

Domain 1

Polypeptide

Intron IntronExon 2 Exon 3

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Modification at both ends

• 1. A 5’ cap is added to the 5’ end of the pre-mRNA as it is transcribed

– Helps the binding of mRNA to the ribosome for translation

– Protects the mRNA from being digested by ribonucleases (enzymes that break down RNAs)

• 2. A Poly A tail is added to the 3’ end of the pre-mRNA at the end of transcription

– Assists in the export of mRNA from the nucleus

– Important for mRNA stability

RNA splicing animation

• http://www.phschool.com/science/biology_place/biocoach/transcription/premrna.html

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

• Translation occurs at the ribosome

• In Eukaryotes transcription and translation are separated by the nucleus

• Transcription: In nucleus

• Translation: In cytoplasm

The information for protein synthesis lies in the genetic code

• Codons-series of genetic information in sequential three letter words of an mRNA molecule

• Specify an amino acid

• Complementary to the corresponding triplet sequence from the DNA molecule it was transcribed

http://en.wikipedia.org/wiki/Genetic_code

Characteristics of the Genetic Code

• A triplet codon could have 64 variations (4 x 4 x 4)

• These codons only determine 20 amino acids

• AUG (methionine) is a start codon which initiates translation

• Stop codons- signal termination of translation

http://chemistry.umeche.maine.edu/CHY431/Code.html

• tRNA links the information contained in each mRNA codon with a specific amino acid

• Two events must take place for the right protein to be made:– A tRNA must chemically read each mRNA codon

correctly

– The tRNA must deliver the amino acid that corresponds to the mRNA codon

Transfer RNAs carry specific amino acids and bind to specific codons

• One specific tRNA molecule for each amino acid

• Three functions of tRNA:– tRNAs bind to particular amino acids

– tRNAs bind to mRNA. The tRNA has a triplet of bases called the anticodon that is complementary to the mRNA codon for the amino acid the mRNA is coding for

–tRNAs interact with ribosomes (ribosomes have several sites on its surface for the tRNA to bind to)

• Each mRNA codon binds to just one tRNA species, carrying a specific amino acid

Each tRNA is specifically attached to an amino acid

• A specific amino acid binds to a specific tRNA molecule

Translation occurs at the ribosome

• The ribosome is where all of the protein synthesis (translation) takes place

• The ribosome is made up of two subunits that are made of rRNA and proteins

• The two subunits come together for translation but remain separate when not translating

• On the large subunit of the ribosome there are three sites that the tRNA can bind to

• They designate them the A, P, and E site

• The mRNA and ribosome move in relation to one another and a charged tRNA goes through these sites in order

• Three different sites:

The A (amino site)

• tRNA anticodon binds to the mRNA codon

• Lines up the correct amino acid to be added to the growing polypeptide chain

The P (polypeptide) site

• The tRNA adds its amino acid to the polypeptide chain

The E (exit) site

• The tRNA resides before being released from the ribosome

• Goes back to the cytosol to pick up another amino acids and begin the process again

Translation takes place in three steps

• Initiation

• Elongation

• Termination

Initiation

• The Initiation complex consists of a charged tRNA and a small ribosomal subunit both bound by mRNA

• The small subunit then goes along the mRNA strand until it finds the start codon (AUG)

• The tRNA molecule binds and then the large subunit of the ribosome joins the complex

Elongation

• The next tRNA then enters the A site of the large subunit

• The new tRNA’s amino acid then gets bonded to the methionine start codon with a peptide bond

• This is done in two enzyme reactions

• The process keeps going with new tRNA and amino acids

Termination

• The elongation cycle ends when a stop codon enters the A site

• These codons bind a protein called a release factor

• The new completed polypeptide separates from the ribosome

Polysome formation increases the rate of protein synthesis

• Several ribosomes can translate a single mRNA molecule at the same time producing multiple polypeptides

• This is called a polyribosome or polysome

• Cells that make a lot of proteins have many polysomes

Translation Video

http://en.wikipedia.org/wiki/Posttranslational_modification

Signal sequences in proteins direct them to their cellular destinations

• After the protein is made it usually stays in the cytosol and performs its function

• Some proteins contain a signal sequence which is a short stretch of amino acids that indicates where in the cell the polypeptide belongs

• (could go to the nucleus, mitochondria, plastids…etc.)

• The signal sequence binds to a receptor protein at the surface of the organelle

• A channel than forms in the organelle membrane allowing the targeted protein to move into the organelle

17.5 Mutations of one or a few nucleotides can affect protein structure and function

Point mutations confirm the genetic code

• Point mutations can show problems with the genetic code and how the amino acid sequence is affected

• Four different kinds:

1. Silent mutations

• Occur because of the redundancy of the genetic code

• Happens when a mutation causes no change in an amino acid because some codons code for the same amino acid

Silent mutations

http://academic.brooklyn.cuny.edu/biology/bio4fv/page/molecular%20biology/mutation-consequences.html

2. Missense mutations

• Result in a change in the amino acid sequence

• GAU translates into (aspartic acid)

• GUU translates into (Valine)

http://ghr.nlm.nih.gov/handbook/illustrations/missense

3. Nonsense mutations

• Results in a stop codon

• The polypeptide chain would end at the amino acid translated just before the stop codon

Nonsense Mutation

http://ghr.nlm.nih.gov/handbook/illustrations/nonsense

4. Frame-shift mutation

• Result from the insertion or deletion of one or more base pairs within the coding sequence

• This can cause new triplets to be read and an altered sequence of amino acids

Frameshift mutation

New Mutations and Mutagens• Spontaneous mutations can occur during

errors in DNA replication, recombination, or repair

• Mutagens are physical or chemical agents that can cause mutations

• Chemical mutagens fall into a variety of categories

• Most carcinogens (cancer-causing chemicals) are mutagens, and most mutagens are carcinogenic

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What Is a Gene? Revisiting the Question

• The idea of the gene has evolved through the history of genetics

• We have considered a gene as– a discrete unit of inheritance – a region of specific nucleotide sequence in

a chromosome– a DNA sequence that codes for a specific

polypeptide chain

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• A gene can be defined as a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule

© 2017 Pearson Education, Inc.

Wild type

DNA template strandA T G A A G T T T G G C T A A

mRNAProtein Met Lys Phe Gly Stop

Amino end Carboxyl end

(a) Nucleotide-pair substitution

A instead of G

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

U instead of C

A U G A A G U U U G G U U A A

Met Lys Phe Gly Met

(b) Nucleotide-pair insertion or deletion

Extra A

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

Extra U

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

StopStopSilent

T instead of C

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

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

Frameshift (1 nucleotide-pair insertion)

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

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

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

Met Lys Leu Ala

3′

A instead of G

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

Met Lys Phe Ser StopMissense

A instead of T

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

U instead of A

A U G U A G U U U G G U U A A

Met Stop

Frameshift (1 nucleotide-pair deletion)

T T C

3′5′

5′

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

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

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

Met Phe Gly Stop

Nonsense 3 nucleotide-pair deletion

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

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

3′5′

3′5′

3′5′

3′5′

3′5′ 3′

5′

3′5′

3′5′

3′5′

3′5′

3′5′

3′5′

3′5′

5′ 3′

5′ 3′

5′ 3′

5′ 3′

5′ 3′

5′

3′

© 2017 Pearson Education, Inc.