Chapter 17RNA and Protein Synthesis

Transcription and Translation

• http://www.dnatube.com/video/3059/DNA-Transcription-and-Protein-Assembly

Overview• Nucleotide sequences responsible for

information on DNA• The DNA inherited yields specific traits by

dictating the synthesis of proteins• Proteins are the links between genotype and

phenotype• Gene expression, the process by which DNA

directs protein synthesis, includes two stages: transcription and translation

History of Gene Expression

• 1909 – Archibald Garrod– First to say genes determine phenotypes through

enzymes that catalyze specific chemical reactions– “inborn errors of metabolism” - symptoms of an

inherited disease reflect an inability to synthesize a certain enzyme

History of Gene Expression

• George Beadle and Edward Tatum – exposed bread mold to X-rays

• mutants were unable to survive on minimal medium – unable to synthesize certain molecules

– Identified three classes of arginine-deficient mutants, each lacking a different enzyme necessary for synthesizing arginine

• One gene–one enzyme hypothesis: states that each gene dictates production of a specific enzyme

More Recent History of Gene Expression

• Many proteins are composed of several polypeptides, each of which has its own gene

• Beadle and Tatum’s hypothesis is now restated as the one gene–one polypeptide hypothesis

The Structure of RNA

• single stranded• working copy of one

gene– Disposable

• RNA is the intermediate between genes and the proteins for which they code

The Structure of RNA

• one gene from DNA can produce many strands of RNA

• instead of thymine (T), uracil (U)

• RNA has A, U, C, G

Transcription and Translation• Transcription is the synthesis of RNA under

the direction of DNA– nucleus

• Transcription produces messenger RNA (mRNA)

• Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA– cytoplasm

• Ribosomes are the sites of translation

Transcription and Translation Overview

• In prokaryotes, mRNA produced by transcription is immediately translated without more processing

• In a eukaryotic cell, the nuclear envelope separates transcription from translation

• Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA

Fig. 17-3

TRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

(a) Bacterial cell

Nuclearenvelope

TRANSCRIPTION

RNA PROCESSINGPre-mRNA

DNA

mRNA

TRANSLATION Ribosome

Polypeptide

(b) Eukaryotic cell

Fig. 17-3a-1

TRANSCRIPTIONDNA

mRNA

(a) Bacterial cell

Fig. 17-3a-2

(a) Bacterial cell

TRANSCRIPTIONDNA

mRNA

TRANSLATIONRibosome

Polypeptide

Fig. 17-3b-1

(b) Eukaryotic cell

TRANSCRIPTION

Nuclearenvelope

DNA

Pre-mRNA

Fig. 17-3b-2

(b) Eukaryotic cell

TRANSCRIPTION

Nuclearenvelope

DNA

Pre-mRNARNA PROCESSING

mRNA

Fig. 17-3b-3

(b) Eukaryotic cell

TRANSCRIPTION

Nuclearenvelope

DNA

Pre-mRNARNA PROCESSING

mRNA

TRANSLATION Ribosome

Polypeptide

17.2 Transcription

DNA RNA

Types of RNA

• three types– messenger RNA

(mRNA)– ribosomal RNA (rRNA)– transfer RNA (tRNA)

Transcription• requires enzyme RNA

polymerase• RNA polymerase binds to

DNA and separates the strands – Similar to DNA replication

Transcription

• RNA polymerase uses one strand of DNA as a template to make RNA

• RNA is made in the 5’ – 3’ direction– How is the DNA

read?

Transcription

• The DNA sequence where RNA polymerase attaches is called the promoter

• In bacteria, the sequence signaling the end of transcription is called the terminator

• The stretch of DNA that is transcribed is called a transcription unit

Fig. 17-7

Promoter Transcription unit

Start pointDNA

RNA polymerase

5533

Initiation1

2

3

5533

UnwoundDNA

RNAtranscript

Template strandof DNA

Elongation

RewoundDNA

5

55

5

5

33

3

3

RNAtranscript

Termination

5533

35Completed RNA transcript

Newly madeRNA

Templatestrand of DNA

Direction oftranscription(“downstream”)

3 end

RNApolymerase

RNA nucleotides

Nontemplatestrand of DNA

Elongation

Fig. 17-7a-1Promoter Transcription unit

DNAStart point

RNA polymerase

553

3

Fig. 17-7a-2Promoter Transcription unit

DNAStart point

RNA polymerase

553

3

Initiation

33

1

RNAtranscript

5 5

UnwoundDNA

Template strandof DNA

Fig. 17-7a-3Promoter Transcription unit

DNAStart point

RNA polymerase

553

3

Initiation

33

1

RNAtranscript

5 5

UnwoundDNA

Template strandof DNA

2 Elongation

RewoundDNA

5

5 5 3 3 3

RNAtranscript

Fig. 17-7a-4Promoter Transcription unit

DNAStart point

RNA polymerase

553

3

Initiation

33

1

RNAtranscript

5 5

UnwoundDNA

Template strandof DNA

2 Elongation

RewoundDNA

5

5 5 3 3 3

RNAtranscript

3 Termination

5

5 5 33

3Completed RNA transcript

Fig. 17-7b

Elongation

RNApolymerase

Nontemplatestrand of DNA

RNA nucleotides

3 end

Direction oftranscription(“downstream”) Template

strand of DNA

Newly madeRNA

3

5

5

Transcription Overview

• http://www.youtube.com/watch?v=WsofH466lqk

Three Stages of Transcription

• Initiation

• Elongation

• Termination

Initiation

• Signaled by promoters• Transcription factors mediate the binding of

RNA polymerase and the initiation of transcription

• Transcription initiation complex: all transcription factors and RNA polymerase II bound to a promoter

• A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes

Fig. 17-8A eukaryotic promoterincludes a TATA box

3

1

2

3

Promoter

TATA box Start point

Template

TemplateDNA strand

535

Transcriptionfactors

Several transcription factors mustbind to the DNA before RNApolymerase II can do so.

5533

Additional transcription factors bind tothe DNA along with RNA polymerase II,forming the transcription initiation complex.

RNA polymerase IITranscription factors

55 53

3

RNA transcript

Transcription initiation complex

Elongation

• RNA polymerase untwists the DNA, 10 to 20 bases at a time

• Transcription progresses at a rate of 40 nucleotides per second in eukaryotes

• A gene can be transcribed simultaneously by several RNA polymerases

Termination

• Bacteria: the polymerase stops transcription at the end of the terminator

• Eukaryotes: the polymerase continues transcription after the pre-mRNA is cleaved from the growing RNA chain– Pre-mRNA will be cleaved when the RNA

polymerase II transcribes a polyadenylation sequence (AAUAAA on the pre-mRNA)

– the polymerase eventually falls off the DNA

17.3 RNA Processing

RNA Processing

• Enzymes in the eukaryotic nucleus modify pre-mRNA

• During RNA processing, both ends of the primary transcript are usually altered

• Some interior parts of the molecule are cut out, and the other parts spliced together

RNA Modifications: the ends

• Each end of a pre-mRNA molecule is modified in a particular way:– The 5’ end receives a modified guanine nucleotide

5’ cap– The 3’ end gets a poly-A tail (made of 50-250 more

adenine nucleotides)

RNA Modifications: the ends

• These modifications share several functions:– Facilitate the export of mRNA– Protection of the mRNA from hydrolytic enzymes– They help ribosomes attach to the 5’ end

Fig. 17-9

Protein-coding segment Polyadenylation signal3

3 UTR5 UTR

5

5 Cap Start codon Stop codon Poly-A tail

G P PP AAUAAA AAA AAA…

RNA Splicing

• Noncoding stretches of nucleotides that lie between coding regions• intervening sequences or introns

• Exons: eventually expressed, translated into amino acid sequences

• RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence

RNA Processing

• RNA will copy both introns and exons from the DNA– Introns need to be cut out

to make a working piece of RNA

• Remaining pieces of exons will be spliced together to form the final mRNA sequence

Fig. 17-10

Pre-mRNA

mRNA

Codingsegment

Introns cut out andexons spliced together

5 Cap

Exon Intron5

1 30 31 104

Exon Intron

105

Exon

146

3Poly-A tail

Poly-A tail5 Cap

5 UTR 3 UTR1 146

• In some cases, RNA splicing is carried out by spliceosomes

• Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Fig. 17-11-1RNA transcript (pre-mRNA)

Exon 1 Exon 2Intron

ProteinsnRNA

snRNPs

Otherproteins

5

Fig. 17-11-2RNA transcript (pre-mRNA)

Exon 1 Exon 2Intron

ProteinsnRNA

snRNPs

Otherproteins

5

5

Spliceosome

Fig. 17-11-3RNA transcript (pre-mRNA)

Exon 1 Exon 2Intron

ProteinsnRNA

snRNPs

Otherproteins

5

5

Spliceosome

Spliceosomecomponents

Cut-outintronmRNA

Exon 1 Exon 25

Ribozymes

• Ribozymes are catalytic RNA molecules that function as enzymes and can splice RNA

• The discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

RNA can function as an enzyme

• It can form a three-dimensional structure because of its ability to base pair with itself

• Some bases in RNA contain functional groups• RNA may hydrogen-bond with other nucleic

acid molecules

Alternative RNA Splicing

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

exons during RNA splicing• Number of different proteins an organism can

produce is much greater than its number of genes

Protein Domains

• 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– Protein will fold differently = act differently

Reading the Genetic Code

The Genetic Code

• Genetic code, or language of mRNA instructions, is read three letters at a time

The Genetic Code

• Each three letter sequence, or “word”, codes for one amino acid• these three letter words are known as codons• Codons are found on the mRNA

• The protein is determined by the order of each amino acid in the sequence

The Genetic Code• There are 64 possible

codons• One codon, AUG, is known

as the “start” codon for protein synthesis

• There are also three possible sequences that are called “stop” codons– Show where polypeptide ends

Translation

Fig. 17-13

Polypeptide

Ribosome

Aminoacids

tRNA withamino acidattached

tRNA

Anticodon

TrpPhe Gly

Codons 35

mRNA

Translation

• Making a protein from a strand of mRNA

tRNA

• Consists of a single RNA strand that is only about 80 nucleotides long

• Flattened into one plane to reveal its base pairing, a tRNA molecule looks like a cloverleaf

Fig. 17-14

Amino acidattachment site

3

5

Hydrogenbonds

Anticodon

(a) Two-dimensional structure

Amino acidattachment site

5

3

Hydrogenbonds

3 5AnticodonAnticodon

(c) Symbol used in this book(b) Three-dimensional structure

In order to have accurate translation…

• First: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase

• Second: a correct match between the tRNA anticodon and an mRNA codon

• Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon

Fig. 17-15-1

Amino acid Aminoacyl-tRNAsynthetase (enzyme)

ATP

AdenosineP P P

Fig. 17-15-2

Amino acid Aminoacyl-tRNAsynthetase (enzyme)

ATP

AdenosineP P P

AdenosineP

PP i

PPi

i

Fig. 17-15-3

Amino acid Aminoacyl-tRNAsynthetase (enzyme)

ATP

AdenosineP P P

AdenosineP

PP i

PPi

i

tRNA

tRNA

Aminoacyl-tRNAsynthetase

Computer model

AMPAdenosineP

Fig. 17-15-4

Amino acid Aminoacyl-tRNAsynthetase (enzyme)

ATP

AdenosineP P P

AdenosineP

PP i

PPi

i

tRNA

tRNA

Aminoacyl-tRNAsynthetase

Computer model

AMPAdenosineP

Aminoacyl-tRNA(“charged tRNA”)

Ribosomes

• Ribosomal RNA (rRNA)• Large subunit• Small subunit

Fig. 17-16Growingpolypeptide Exit tunnel

Largesubunit

Smallsubunit

tRNAmolecules

E P A

mRNA5 3

(a) Computer model of functioning ribosome

P site (Peptidyl-tRNAbinding site)

E site(Exit site)

A site (Aminoacyl-tRNA binding site)

E P A Largesubunit

mRNAbinding site

Smallsubunit

(b) Schematic model showing binding sites

Amino end Growing polypeptide

Next amino acidto be added topolypeptide chain

mRNAtRNAE

3

5 Codons

(c) Schematic model with mRNA and tRNA

Fig. 17-16b

P site (Peptidyl-tRNAbinding site) A site (Aminoacyl-

tRNA binding site)E site(Exit site)

mRNAbinding site

Largesubunit

Smallsubunit

(b) Schematic model showing binding sites

Next amino acidto be added topolypeptide chain

Amino end Growing polypeptide

mRNAtRNA

E P A

E

Codons

(c) Schematic model with mRNA and tRNA

5

3

Ribosomes

• A ribosome has three binding sites for tRNA:– The P site (peptidyl-tRNA binding site) holds the

tRNA that carries the growing polypeptide chain– The A site (Aminoacyl-tRNA binding site) holds

the tRNA that carries the next amino acid to be added to the chain

– The E site (exit site) is where discharged tRNAs leave the ribosome

Three Parts of Translation

• Initiation• Elongation• Termination

Steps of Translation

1. mRNA attaches to a ribosome

• a small ribosomal subunit binds with mRNA and a special initiator tRNA

• small subunit moves along the mRNA until it reaches the start codon (AUG)

• initiation factors bring in the large subunit that completes the translation initiation complex

Fig. 17-17

3355U

UA

ACGMet

GTP GDPInitiator

tRNA

mRNA

5 3Start codon

mRNA binding site

Smallribosomalsubunit

5

P site

Translation initiation complex

3

E A

Met

Largeribosomalsubunit

Elongation

• Amino acids are added one by one to the preceding amino acid

• Each addition involves proteins called elongation factors and occurs in three steps:– codon recognition

– peptide bond formation

– Translocation

Elongation: Codon Recognition

2. tRNA brings the correct amino acid to the ribosome

• each tRNA carries only one type of amino acid– the anticodon sequence

is complimentary to the codon

Elongation: Peptide Bond Formation

3. Ribosome forms a peptide bond between the amino acids (forming a protein)

Elongation: Translocation

4. The tRNA from the first amino acid is then released from the ribosome via the E site

5. Ribosome then moves to the next codon and a new tRNA enters the A site

Translation Continues!

6. Polypeptide chain grows until the ribosome reaches a “stop” codon

Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome

Translation Complete!

7. The A site accepts a protein called a release factor

The release factor causes the addition of a water molecule instead of an amino acid

Releases the polypeptide, and the translation assembly then comes apart

Fig. 17-19-1

Releasefactor

3

5Stop codon(UAG, UAA, or UGA)

Fig. 17-19-2

Releasefactor

3

5Stop codon(UAG, UAA, or UGA)

5

3

2

Freepolypeptide

2 GDP

GTP

Fig. 17-19-3

Releasefactor

3

5Stop codon(UAG, UAA, or UGA)

5

3

2

Freepolypeptide

2 GDP

GTP

5

3

Polyribosomes

• Many ribosomes translate a single mRNA simultaneously, forming a polyribosome (or polysome)

• Polyribosomes enable a cell to make many copies of a polypeptide very quickly

Fig. 17-20

Growingpolypeptides

Completedpolypeptide

Incomingribosomalsubunits

Start ofmRNA(5 end)

Polyribosome

End ofmRNA(3 end)

(a)

Ribosomes

mRNA

(b) 0.1 µm

• Polypeptide chains are modified after translation

• Completed proteins are targeted to specific sites in the cell

• During and after synthesis, a polypeptide chain spontaneously coils and folds into its three-dimensional shape

• Proteins may also require post-translational modifications before doing their job

• Some polypeptides are activated by enzymes that cleave them

• Other polypeptides come together to form the subunits of a protein

Ribosomes• Free ribosomes: in cytosol• Bound ribosomes: on surface of ER– Make proteins to be excreted from the cell– All transcription occurs in the cytoplasm• Polypeptides destined for the ER or for secretion are

marked by a signal peptide • A signal-recognition particle (SRP) binds to the signal

peptide• The SRP brings the signal peptide and its ribosome to the ER

Mutations

• Mutations are changes in the genetic material of a cell or virus

• Point mutations are chemical changes in just one base pair of a gene– Substitution– Insertion or Deletion

Effects of Substitutions

• Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code

• Missense mutations still code for an amino acid, but not necessarily the right amino acid

• Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein

Effects of Insertions and Deletions

• More harmful than substitutions• Insertion or deletion of nucleotides may alter

the reading frame, producing a frameshift mutation

Mutagens

• Spontaneous mutations can occur during DNA replication, recombination, or repair

• Mutagens are physical or chemical agents that can cause mutations

Gene Expression in Archae, Bacteria, Eukaryotes

• Use your text book to compare and contrast the cellular processes of gene expression in archaea, bacteria and eukarya

Genes and Proteins

• genes code for proteins which are what carry out expression of these genes

• proteins code for enzymes which cause certain reactions to take place– these reactions are what cause traits!

Protein Structure

Protein Structure

• Primary (1°)structure: amino acid sequence from translation

• Secondary (2°) structure: alpha (α) helix and beta (β) sheets

Protein Structure

• Tertiary (3º) structure: three dimensional structure– Each protein has its own

unique tertiary structure

Protein Structure• Quaternary (4°)

structure: multiple tertiary structures form a complex

• The protein will change confirmation (shape) if not being used