Chapter 12:RNA and Protein Synthesis

Gene Expression – How DNA affects Phenotype

DNA proteins phenotype

2 steps

– DNA mRNA

• Translation – mRNA protein

Fig. 17-3b-3

(b) Eukaryotic cell

TRANSCRIPTION

Nuclearenvelope

DNA

Pre-mRNARNA PROCESSING

mRNA

TRANSLATION Ribosome

Polypeptide

Fig. 17-4

DNAmolecule

Gene 1

Gene 2

Gene 3

DNAtemplatestrand

TRANSCRIPTION

TRANSLATION

mRNA

Protein

Codon

Amino acid

RNA – ribonucleic acid

• Single stranded• nucleotides

– Ribose– Phosphate– AUCG

• U = Uracil

RNA• Transcription (DNA template mRNA)• 3 types

– Ribosomal RNA (rRNA)– Transfer RNA (tRNA)– Messenger RNA (mRNA)

• Made from DNA – DNA-dependent RNA polymerases• Make RNA from DNA in 5’ 3’ direction, DNA read

3’5’• DNA template and new RNA are antiparallel

• Upstream – toward 5’ of mRNA OR 3’ of DNA• Downstream – toward 3’ of RNA OR 5’ of DNA

Transcription• 1. RNA polymerase binds to DNA at Promoter

– Promoter not transcribed– RNA polymerase passes promoter; begins

transcribing DNA– No primer required

• 2. RNA nucleotides added to 3’ end of RNA– 1st RNA (5’ end) keeps triphosphate– RNA nucleotides added lose 2 P and 3rd P becomes

part of sugar-phosphate backbone– Last RNA nucleotide – exposed 3’ OH

Transcription

• 3. Termination – Stop sequence at end of gene

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

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

Resulting mRNA• Leader sequence –

– Noncoding– Made because RNA polymerase starts transcription

well upstream of coding sequence

• Coding sequence – – Codes for proteins

• Termination or stop codon– End of coding sequence– UAA, UGA, UAG– Don’t code for AA; specify end of protein– Followed by noncoding 3’ trailing sequences

Transcription

Posttranscriptional modification and processing

• Precursor mRNA = original mRNA transcript (pre-mRNA)

• Begins – RNA transcript is 20-30 nucleotides long

• Enzymes add cap to 5’ end of mRNA– Need cap for eukaryotic ribosome to bind– May protect from degradation

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…

• Polyadenylated (poly-A) tail gets added– 3’ end– When transcript complete, enzymes in nucleus

recognize polyadenylation signal and cut mRNA at that site

– 100-250 adenine nucleotides are added by enzymes to 3’ end

– May help• Export mRNA from nucleus, fight degradation, make

translation initiation more efficient

Take out noncoding sequences

• Interrupted coding sequences = long sequences of bases in the protein-coding sequences of the gene that do not code for AA in the final protein product INTRONS (noncoding regions)

• EXONS – (expressed sequences) – part of the protein-coding sequence

• Introns are removed and splice exons together continuous coding sequence

• Small nuclear ribonucleoprotein complexes (snRNPs) – bind to introns and catalyze the excision and splicing reactions

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

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

Exon 1 Exon 2Intron

ProteinsnRNA

snRNPs

Otherproteins

5

5

Spliceosome

Spliceosomecomponents

Cut-outintronmRNA

Exon 1 Exon 25

mRNA processing

Fig. 17-13

Polypeptide

Ribosome

Aminoacids

tRNA withamino acidattached

tRNA

Anticodon

TrpPhe Gly

Codons 35

mRNA

Translation: mRNA AA (protein)

• Codon – sequence of 3 consecutive bases in mRNA (triplet code); specify 1 AA

• Transfer RNA (tRNA) – connect AA and mRNA; link with specific AA

• Anticodon – sequence of 3 bases on tRNA; H bonds with mRNA codon by base-pairing rules

• Aminoacyl-tRNA synthetase – enzyme that links amino acids to tRNAs

• Make aminoacyl-tRNAs (can bind to mRNA)

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”)

Properties of tRNA

• 1. anticodon – 3 base triplet complementary to mRNA codon

• 2. must be recognized by specific aminoacyl-tRNA synthetase that adds correct AA

• 3. must have region that serves as attachment site for specific AA specified by anticodon

• 4. must be recognized by ribosomes

tRNA

• Gets folded on itself (base-pairing within tRNA) 3+ loops (unpaired bases)

• 1 of the loops has anticodon• 3’ end – AA binding site• Carboxyl of AA binds to OH tRNA at 3’ end,

leaving amino group on AA to make peptide bond

Fig. 17-14a

Amino acidattachment site

(a) Two-dimensional structure

Hydrogenbonds

Anticodon

3

5

Translation – At Ribosomes• Ribosome

– Made of 2 different subunits (proteins and ribosomal RNA)

– rRNA – no transfer of info, has catalytic functions– Attach to 1 end of mRNA and travel along it,

allowing tRNAs to attach in sequence to mRNA codons

Ribosomes

– mRNA fits in groove between 2 subunits– Holds mRNA, aminoacyl tRNA and growing

polypeptide chain– tRNAs attach to A and P binding sites

• A site – new AA dock; AA form bond with polypeptide chain and tRNA moves to P site

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

3 stages of Translation

• Initiation, repeating cycles of elongation, termination

Initiation • Initiation factors (proteins) attach to small

subunit, allowing it to bind to a special initiator tRNA

• Initiator tRNA is loaded onto small subunit, making initiation complex

• Initiation complex binds to special ribosome-recognition sequences upstream of coding sequences on mRNA (near 5’ end)

• Initiation complex moves along mRNA until it reaches an initiator codon = AUG

• Anticodon of initiator tRNA binds to initiation codon of mRNA

• Large subunit attaches to complex• completed ribosome

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• Addition of AA to A site by base pairing of

anticodon w/ codon • Ribosome moves in 3’ direction along mRNA• Needs energy from GTP• Peptidyl transferase – ribozyme – rRNA component

of large subunit– AA at P site released from its tRNA (in P site)– Peptidyl transferase attaches this AA to aminoacyl-tRNA

at A site– Peptide bond formed – translocation – chain moves to P

site, leaving A site open– GTP for bond, none for transferase

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

Termination

• “Release factors” stop translation• Recognize termination (stop) codons• Release newly-made protein, mRNA and last

tRNA, causing ribosome to dissociate

Fig. 17-19-3

Releasefactor

3

5Stop codon(UAG, UAA, or UGA)

5

3

2

Freepolypeptide

2 GDP

GTP

5

3

Translation

Protein Synthesis:Eukaryotes vs. Prokaryotes

Prokaryotes• mRNA is translated as it is

being transcribed from DNA (no nucleus to exit)

• mRNA used immediately, no further processing

Eukaryotes• mRNA must be transported

to cytoplasm before translation

• Original mRNA transcript must be modified before leaving the nucleus

Fig. 17-3

TRANSCRIPTION

TRANSLATION

DNA

mRNARibosome

Polypeptide

(a) Bacterial cell

Nuclearenvelope

TRANSCRIPTION

RNA PROCESSINGPre-mRNA

DNA

mRNA

TRANSLATION Ribosome

Polypeptide

(b) Eukaryotic cell

Special features of the Genetic Code

• 3 letter combos from 4 bases – specify 64 AA• Nirenberg and Matthaei

– Experimented to determine which AA were coded for by specific mRNA codons

– Ex: UUUUUUUUU… - only found phenylalanine so UUU = phenylalanine

– Found 3 stop codons – specified no AA• UAA, UGA, UAG

Fig. 17-5Second mRNA base

Fir

st

mR

NA

ba

se

(5

e

nd

of

co

do

n)

Th

ird

mR

NA

ba

se

(3

e

nd

of

co

do

n)

The Genetic Code is Universal

• All organisms• Few coding exceptions

– Protozoans – UAA, UGA for glutamine, instead of stop

– Mitochondria - own DNA

Wobble Hypothesis

• 61 codons, but only 40 different tRNAs• tRNA can pair w/ 1+ codon• Francis Crick• 3rd nucleotide of tRNA anticodon (5’ end) may

be capable of forming H bonds w/ more than 1 kind of 3rd nucleotide (3’ end) of codon

Reverse?

• Howard Temin – proposed DNA provirus formed as intermediate in replication of RNA tumor viruses

• RNA-directed DNA polymerase (Reverse transcriptase) – made DNA from RNA template

• Retroviruses – HIV

Mutations • Changes in nucleotide sequence of DNA• Spontaneously during DNA replication, mitosis,

meiosis, or because of mutagens• Low rate of occurrence because of cell’s repair

mechanisms• Provide diversity of genes

– Variation– evolution

• Copied as normal, no greater chance of further mutation (normally)

Base substitution mutation

• Simplest• 1 pair nucleotides changes• From errors in base pairing during replication• Affects transcribed mRNA and polypeptide

Missense mutations

• Base substitutions that result in the replacement of 1 AA by another

• Wide range of effects– Enzyme activity– “silent” – substituted w/ closely related AA,

effects undetectable

Nonsense mutations

• Base substitution that converts an AA-specifying codon to a termination codon

• Usually destroys function of gene product

Frameshift mutations

• 1 or 2 nucleotide pairs are inserted or deleted from the molecule, causing an alteration of the reading frame

• Codons downstream now specify entirely new sequence of AA

• Different effects depends where it happens• Entirely new polypeptide• Stop codon w/in short distance of mutation• Loss enzyme activity (disastrous)

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

Jumping Genes

• (mobile genetic elements, transposable elements, transposons)

• DNA sequence “jumps” to middle of gene, disrupting gene functions and/or activation previously inactive genes

• Genes spontaneously turned on or off• Barbara McClintock – 1950s• Require transposase enzyme

Mutagens

• Mutagens – Agents that cause mutations– Radiation – X, gamma, cosmic, UV rays; chemicals

• Carcinogens – Cause cancer

Fig. 17-25

TRANSCRIPTION

RNA PROCESSING

DNA

RNAtranscript

3

5RNApolymerase

Poly-A

Poly-A

RNA transcript(pre-mRNA)

Intron

Exon

NUCLEUS

Aminoacyl-tRNAsynthetase

AMINO ACID ACTIVATIONAminoacid

tRNACYTOPLASM

Poly-A

Growingpolypeptide

3

Activatedamino acid

mRNA

TRANSLATION

Cap

Ribosomalsubunits

Cap

5

E

P

A

AAnticodon

Ribosome

Codon

E

Fig. 17-UN8