chapter 17 from gene to protein

A A A A

A A A A A

G

G

G

G

G

G G

G

GT T T

T T T T T

T

C C

C C

C C C

C C

The connection

between genes and proteins• A = Adenine = Base• TACCGCCTAA = Sequence of bases on

DNA• The above sequence of bases is arranged,

not by chance but because it specifies a particular gene

From gene protein …overview

• Transcription: Template strand of DNA is copied/transcribed into another sequence of bases (similar language. Instead of T, U [Uracil] RNA [mRNA])

AUGGCGGAUU This is complimentary,

but not identical to the template DNA

strand

From gene protein …overview

• Translation: Translate DNA/RNA language into a whole new language—amino acid lingo

Translated into triplet code. Each 3 bases is a codon.AUG GCG GAU U….

Each codon specifies a particular amino acid (20

different amino acids)

AUG = “start codon”—specifies amino

acid methionine

AA + AA + AA + AA = polypeptide

polypeptide + polypeptide = protein!!!

• DNA RNA Protein– It’s these proteins

that make us look different

– On the flip side, it’s also a common thread between all living organisms

• DNA is the universal life language– Organisms can

express foreign DNA (E. Coli & insulin; tobacco plant & firefly gene)

Fig. 17-6

(a) Tobacco plant expressing a firefly gene

(b) Pig expressing a jellyfish gene

• Protein Synthesis is slightly different in Prokaryotes–No nucleus—

processes are not separated by space and time as they are in Eukaryotes

BioFlix: Protein Synthesis

From gene protein … the details

DNA RNAA piece of RNA is made or

copied from the template strand of DNA (transcription unit)

A. Initiation 1. Starts at Promoter—

determines which DNA strand will be template and indicates where to begin

- TATA box (upstream)—specific DNA sequence

2. Transcription factors (proteins) and other proteins help bind RNA polymerase to DNA template strand

I. Transcription

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

transcription in action

Fig. 17-7b

Elongation

RNApolymerase

Nontemplatestrand of DNA

RNA nucleotides

3 end

Direction oftranscription(“downstream”) Template

strand of DNA

Newly madeRNA

3

5

5

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

From gene protein … the details

Transcription factors + RNA polymerase

+ promoter(all bound together)

= Transcription Initiation Complex

RNA polymerase can unwind DNA strand and add RNA nucleotides to 3’ end.

B. Elongation RNA polymerase adds

RNA nucleotides. DNA is open 10-20 bases—reforms as transcript peels away

- can occur one transcript at a time

- or many RNA polymerases following each other down the DNA transcribing more for the production of more RNA and thus, more protein

From gene protein … the details

C. Termination RNA polymerase transcribes a DNA sequence called

the Terminator Eukaryotes – RNA polymerase continues for about

10-35 more bases before transcript separates from RNA polymerase

Prokaryotes – Stop immediately after terminator

From gene protein … the details

Step one in protein synthesisTranscription

Transcription intro

RNA ProcessingIn Eukaryotes – the initial transcript (primary transcript or

pre- mRNA)must be modified prior to leaving the nucleus. it becomes finished mRNA.

1. 5’ end is capped (5’ cap) with a modified guanine nucleotide- protects mRNA from degradation- notifies ribosome where to attach (5’ end)- Facilitates export from the nucleus

2. Poly A tail added to 3’ end (30-200 adenines)- inhibits degradation- helps ribosomes attach to the 5’ end- facilitates export from the nucleus

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 Processing

3. RNA splicing Original transcript may have 8000 nucleotides – only need about

1200. (avg. polypeptide is 400 amino acids long) – must cut out unneeded RNA- intervening sequences / introns = non-coding sequences of nucleotides- exons = coding regions – will be expressed

*exception- leader and trailer regions – aren’t coding sequences, but aren’t cut out.

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

RNA ProcessingSmall nuclear ribonucleoproteins

(snRNPs or “snurps”) recognize specific sites flanking introns

SnRNPs + additional proteins Spliceosome

1. Spliceosome interacts with splice site

2. Cuts out intron 3. Releases intron 4. Joins exons together

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; act as enzymes – can splice RNA

• Their discovery disproved the idea that all enzymes are proteins

• Three properties of RNA enable it to function as an enzyme– It can form 3-D 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

**Why introns…….• - have sequences that

control gene activity• - can regulate passage of

mRNA into cytoplasm• - genes can code for 2+

different proteins based on what’s left in and what’s not

•separate exons which may code for different parts of a protein•Organisms can produce more proteins than there are genes•increase chance of crossing over & recombination = increase variation•Exon shuffling may result in the evolution of new proteins

II. TranslationRNA Protein or Codons Amino Acids**Very specific sequences =

DNA RNA amino acids = primary structure

Primary structure determines secondary through quaternary (protein from and function)

DNA TAC GCC CAT

RNA AUG CGG GUA

Amino Acid MET (start)

ARG VAL

MET + ARG + VAL = Polypeptide

Translation and tRNA• tRNA – carries

specific amino acids to site of protein synthesis. How does it know what order to go in?

• Anticodon at the bottom of tRNA is complimentary to a particular mRNA codon (H bond)

• - If mRNA codon is AUG then anticodon is UAC

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

Translation and tRNA• 61 mRNA codons –

only 45 tRNAs – complimentary binding on 3rd nucleotide isn’t strict= wobble

• - Some have inosine (modified base)– can bind with any base

II. Translation cont..Before tRNA can

shuttle over amino acids, it needs to have the right amino acid attached to it

- Amino acids are joined to correct tRNA by enzyme aminoacyl-tRNA synthetase

- 20 different enzymes for each amino acid

Heading off to the Ribosomes!

• mRNA is made and modified

• tRNA has a correct amino acid…all we need now are ribosomes and then a polypeptide can be made!!!!

Heading to the ribosomes….

~Ribosomes – sites of proteins synthesis; join tRNA and mRNA

- consist of a large and small subunit

- made of rRNA and protein

- 3 main sitesBinding site – where mRNA binds1. P site (peptidyl tRNA) – holds tRNA with growing polypeptide chain2. A site (aminoacyl tRNA) – holds tRNA with new, incoming amino acid3. E site (exit) – empty tRNAs leave from here

Fig. 17-16bP 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

Fig. 17-16a

Growingpolypeptide Exit tunnel

tRNAmolecules

Largesubunit

Smallsubunit

(a) Computer model of functioning ribosome

mRNA

E P A

5 3

Process of TranslationA. Initiation

1. Small ribosomal subunit attaches onto leader sequence of mRNA (5’ end), 5’ cap “tells” ribosome where to bind2. Downstream – AUG = start translation3. tRNA with methionine amino acid attaches to initiation codon AUG – tRNA is in the P site. *All of these together (initiation complex) triggers the attachment of the large ribosomal subunit- initiation factors = proteins that bring them all together

Process of TranslationB. ElongationAmino acids are added and connected with the help

of proteins, elongation factors1.Codon recognition

-Incoming tRNA to A site -Anti-codon + codon complementarily – H bonding2.Peptide bond formation -rRNA molecule in ribosome (ribozyme) – catalyzes the formation of a peptide bond -bond forms between last amino acid of pp chain in the p site and new amino acid in a site -tRNA from p site becomes detached from polypeptide chain

3. Translocation

Fig. 17-18-1

Amino endof polypeptide

mRNA

5

3E

Psite

Asite

Fig. 17-18-2

Amino endof polypeptide

mRNA

5

3E

Psite

Asite

GTP

GDP

E

P A

Fig. 17-18-3

Amino endof polypeptide

mRNA

5

3E

Psite

Asite

GTP

GDP

E

P A

E

P A

Fig. 17-18-4

Amino endof polypeptide

mRNA

5

3E

Psite

Asite

GTP

GDP

E

P A

E

P A

GDPGTP

Ribosome ready fornext aminoacyl tRNA

E

P A

Translation

Process of Translation

C. Termination-Stop codon in a site (UAA, UAG, UGA)-Protein release factor binds to stop codon in A site

Adds water to pp chain – hydrolysis between pp chain and tRNA in P site-Polypeptide chain detaches-Subunits and mRNA come 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

32

Freepolypeptide

2 GDP

GTP

Fig. 17-19-3

Releasefactor

3

5Stop codon(UAG, UAA, or UGA)

5

32

Freepolypeptide

2 GDP

GTP

5

3

Fig. 17-13

Polypeptide

Ribosome

Aminoacids

tRNA withamino acidattached

tRNA

Anticodon

Trp

Phe Gly

Codons 35

mRNA

Translation in Action

And…. ACTION!!

While gene expression differs among the domains of life, the concept of a gene is universal

• Archaea are prokaryotes, but share many features of gene expression with eukaryotes

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

Comparing Gene Expression in Bacteria, Archaea, and Eukarya

• Bacteria and eukarya differ in – their RNA polymerases– termination of transcription– ribosomes

• Archaea & eukarya are simlilar in these respects

• Bacteria simultaneously transcribe & translate the same gene

• In eukarya, transcription & translation are separated (Why?)

• In archaea, transcription and translation are likely coupled

Variations on translation:• Single transcribed mRNA can be used to

make many polypeptides at the same time = polyribosomes

• Bacterial Cells – Translate while transcribing

Fig. 17-24RNA polymerase

DNA

Polyribosome

mRNA

0.25 µmDirection oftranscription

DNA

RNApolymerase

Polyribosome

Polypeptide(amino end)

Ribosome

mRNA (5 end)

Fig. 17-20

Growingpolypeptides

Completedpolypeptide

Incomingribosomalsubunits

Start ofmRNA(5 end)

Polyribosome

End ofmRNA(3 end)

(a)

Ribosomes

mRNA

(b) 0.1 µm

• Free=proteins for cell – remain in cytosol• Bound= proteins of endomembrane system – destined for

export• Translation begins in cytosol• Proteins destined for export have a signal peptide (20 AA

at leading end)• Signal recognition particle (SRP) recognizes signal peptide

SRP brings ribosome to ER for the remainder of protein synthesis

-Free ribosomes vs. bound

Fig. 17-21

Ribosome

mRNA

Signalpeptide

Signal-recognitionparticle (SRP)

CYTOSOL Translocationcomplex

SRPreceptorprotein

ER LUMEN

Signalpeptideremoved

ERmembrane

Protein

Translation in action! More fun than the movies!!!

Mistakes• Mistakes made on

DNA can be good or bad

• Bad: wrong bases=wrong amino acid = wrong protein=lethal

• Good: mutation by accident can lead to advantageous proteins = (lead to variations)

Fig. 17-22

Wild-type hemoglobin DNA

mRNA

Mutant hemoglobin DNA

mRNA

33

3

3

3

3

55

5

55

5

C CT T TTG GA A AA

A A AGG U

Normal hemoglobin Sickle-cell hemoglobin

Glu Val

Point Mutation – Mutation on a single nucleotide

Point mutations – mutations made on the DNA level

1.Substitutions:replacement of one nucleotide

-some are silent – may code for the same amino acid

-some can change aa, but the particular substituted amino acid may not effect quaternary structure

-missense mutation –still codes for an amino acid, but it’s the wrong one

-nonsense mutation – can change an amino acid into a stop codon – short protein

Ex: Sickle Cell Anemia

2.Insertions / Deletions -Frameshift mutation – due to the addition or deletion of a nucleotide, the amino acids making up codons are grouped improperly

(codes are read in 3’s)

THE FAT CAT SAT insert a letter, delete a

letter

All of the above are spontaneous

mutations. There are also mutations

caused by mutagens

Fig. 17-23a

Wild type

3DNA templatestrand

3

355

5mRNA

Protein

Amino end

Stop

Carboxyl end

A instead of G

33

3

U instead of C

55

5

Stop

Silent (no effect on amino acid sequence)

Fig. 17-23b

Wild type

DNA templatestrand

35

mRNA

Protein

5

Amino end

Stop

Carboxyl end

53

3

T instead of C

A instead of G

33

3

5

5

5

Stop

Missense

Fig. 17-23cWild type

DNA templatestrand

35

mRNA

Protein

5

Amino end

Stop

Carboxyl end

53

3

A instead of T

U instead of A

33

3

5

5

5

Stop

Nonsense

Fig. 17-23d

Wild type

DNA templatestrand

35

mRNA

Protein

5

Amino end

Stop

Carboxyl end

53

3

Extra A

Extra U

33

3

5

5

5

Stop

Frameshift causing immediate nonsense (1 base-pair insertion)

Fig. 17-23e

Wild type

DNA templatestrand

35

mRNA

Protein

5

Amino end

Stop

Carboxyl end

53

3

missing

missing

33

3

5

5

5

Frameshift causing extensive missense (1 base-pair deletion)

Fig. 17-23fWild type

DNA templatestrand

35

mRNA

Protein

5

Amino end

Stop

Carboxyl end

53

3

missing

missing

33

3

5

5

5

No frameshift, but one amino acid missing (3 base-pair deletion)

Stop

Fig. 17-23Wild-type

3DNA template strand

5

5

53

3

Stop

Carboxyl endAmino end

Protein

mRNA

33

3

55

5

A instead of G

U instead of C

Silent (no effect on amino acid sequence)

Stop

T instead of C

33

3

55

5

A instead of G

Stop

Missense

A instead of T

U instead of A

33

3

5

5

5

Stop

Nonsense No frameshift, but one amino acid missing (3 base-pair deletion)

Frameshift causing extensive missense (1 base-pair deletion)

Frameshift causing immediate nonsense (1 base-pair insertion)

5

5

533

3

Stop

missing

missing

3

3

3

5

55

missing

missing

Stop

5

5533

3

Extra U

Extra A

(a) Base-pair substitution (b) Base-pair insertion or deletion

You should now be able to:

1. Describe the contributions made by Garrod, Beadle, and Tatum to our understanding of the relationship between genes and enzymes

2. Briefly explain how information flows from gene to protein

3. Compare transcription and translation in bacteria and eukaryotes

4. Explain what it means to say that the genetic code is redundant and unambiguous

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

5. Include the following terms in a description of transcription: mRNA, RNA polymerase, the promoter, the terminator, the transcription unit, initiation, elongation, termination, and introns

6. Include the following terms in a description of translation: tRNA, wobble, ribosomes, initiation, elongation, and termination

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