Protein Synthesis

(1)

Protein Synthesis

 Chapter 17~

From Gene to

Protein

(2)

Protein Synthesis: overview



One gene-one enzyme hypothesis (Beadle and Tatum) Mutated mold and identified faulty enzymatic

pathways with differential media



Not all proteins are enzymes: keratin



One gene-one polypeptide (protein) hypothesis



Latest: one gene, 3500 proteins (alternative RNA splicing)



Humans possess about 30,000 genes and make about 100,000 proteins



Transcription: synthesis of RNA under the direction of DNA (mRNA)



Translation: actual synthesis of a

polypeptide under the direction of

mRNA

(3)

The Triplet Code



The genetic instructions for a polypeptide chain are ‘written’ in the DNA as a series of 3-nucleotide ‘words’



Codons



‘U’ (uracil) replaces ‘T’

in RNA

(4)

64 Codons code for 20 AA

 Degenerate code: more than 1 codon codes for the same AA

 ID of the 3rd nucleotide in many codons is not

important:

“Wobble

Hypothesis”

Second mRNA base

U C A G

U

C

A

G

UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG

Met or start

Phe Leu

Leu

lle

Val

UCU UCC UCA UCG

CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG

Ser

Pro

Thr

Ala

UAU UAC

UGU

Tyr UGC Cys

CAU CAC CAA CAG

CGU CGC CGA CGG AAU

AAC AAA AAG

AGU AGC AGA

AGG GAU

GAC GAA GAG

GGU GGC GGA GGG UGG UAA

UAG Stop

Stop UGA Stop Trp His

Gln

Asn Lys

Asp

Arg

Ser Arg

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

First mRNA base (5 end) Third mRNA base (3 end)

Glu

More freedom in pairing up: Inosine, modified adenine,

can bond w/ U,C or A

(5)

Transcription, I



RNA polymerase: pries DNA apart and hooks RNA

nucleotides together using the DNA code.



Eukaryotes use RNA poly II for proteins and I & III for other RNA’s



Promoter region on DNA:

where RNA polymerase

attaches and where initiation of RNA begins



Terminator region:

sequence that signals the end of transcription

(prokaryotes)



Transcription unit:

stretch of DNA transcribed into an RNA molecule

Figure 17.8

TRANSCRIPTION RNA PROCESSING

TRANSLATION

DNA Pre-mRNA mRNA

Ribosome Polypeptide

T A TA A A A A T A T T T T

TATA box Start point Template

DNA strand

53 3

5

Transcription factors 5

3 3

5 Promoter

53 3

5 5

RNA polymerase II

Transcription factors

RNA transcript Transcription initiation complex

Eukaryotic promoters 1

Several transcription factors

2

Additional transcription factors

3

(6)

Transcription, II



Initiation~ transcription factors mediate the

binding of RNA

polymerase to an initiation sequence (TATA box)

Transcript Initiation Complex is formed.



In Prokaryotes RNA poly works alone



Elongation~ RNA

polymerase II continues unwinding DNA and

adding nucleotides to the 3’ end



Termination~ RNA

polymerase reaches

terminator sequence

(7)

Fundamental Prokaryotic &

Eukaryotic Differences



Types of RNA Polymerase

– One in Prokaryotes, 3 in Eukaryotes



Binding the promoter on DNA

– Pro: RNA polymerase binds alone

– Euk: RNA polymerase plus proteins, called Transcription Factors, form a Transcription Initiation Complex. TATA box unique to Euks.



Terminating Transcription

– Pro: Terminator sequence on DNA causes RNA poly to release – Euk: polyadenylation signal (AAUAAA) serves as a marker to cut

the transcript free, RNA poly does not release for 100’s of nucleotides



Post Transcriptional Modification is Eukaryotic

– 5’ cap, 3’ poly A tail, presence of introns & exons

(8)

mRNA modification



1) 5’ cap: modified guanine; protection; recognition site for ribosomes



2) 3’ tail: poly(A) tail (adenine); protection; recognition;

transport



3) RNA splicing: exons (expressed sequences) kept,introns

(intervening sequences) spliced out; spliceosome

(9)

RNA Splicing or Post

Transcriptional Modification



Spliceosomes

(snRNPs & proteins) recognize the

beginning and end of introns and remove them & join exons



A protozoan, tetrahymena,

contains self-splicing rRNA. A ribozyme (enzymatic RNA)

RNA transcript (pre-mRNA)

Exon 1 Intron Exon 2

Other proteins Protein

snRNA

snRNPs

Spliceosome

Spliceosome components

Cut-out intron mRNA

Exon 1 Exon 2 5

5

1

2

3

(10)

Alternative RNA splicing is how one gene can

result in the formation of several different proteins.

Also domain mixing.



Consider a gene with 3 exons. Possibilties:



Exons 1-2-3



Exons 1-3



Exons 2-3



Domains: Enzymatic protein might have 2

domains - #1 active site,

#2 binds the protein to a membrane. Add to the domain of the active site by keeping an intron?

– Add a domain from another portion of mRNA?

DNA Gene

Exon 1 Intron Exon 2 Intron Exon 3 Transcription

RNA processing

Translation

Domain 3

Domain 1 Domain 2

Polypeptide

(11)

Translation: tRNA Carries Specific AA’s

A specific enzyme called an aminoacyl-tRNA

synthetase (many variations)

– Joins each amino acid to the correct tRNA

ATP

(b) Three-dimensional structure

Symbol used in this book Amino acid attachment site

Hydrogen bonds

Anticodon Anticodon

A AG 5 3

3 5

(c)

After tRNA drops off its AA, it can be re-

acylated.

About 45 different

tRNAs exist

(12)

Ribosomes supply the surface for Translation



Consist of a large and small sub unit that only attach during translation



Made of rRNA & proteins



snoRNA(small nucleolar) enzymatically cleaves rRNA transcript into small & large subunits



Some antibiotics cause prokaryotic ribosomes to innactivate

TRANSCRIPTION

TRANSLATION DNA

mRNA Ribosome

Polypeptide Exit tunnel Growing

polypeptide tRNA

molecules

EP A

Large subunit

Small subunit

mRNA

Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The

eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins.

(a)

5 3

(13)

Translation, I

 mRNA from nucleus is ‘read’ along its

codons by tRNA’s anticodons at the ribosome

 tRNA anticodon

(nucleotide triplet);

amino acid

(14)

Translation, II



rRNA ^site

of mRNA codon & tRNA anticodon coupling



P site ^holds

the tRNA carrying the growing polypeptide chain



A site ^holds

the tRNA carrying the next amino acid to be added to the chain



E site discharged

tRNA’s

(15)

Translation, III



Initiation~ union of mRNA, tRNA, small ribosomal subunit;

followed by large subunit completes the Translation initiation complex.

Intitiation factors (proteins) help bind all together. Requires GTP



Elongation~

– •codon recognition (GTP)

•peptide bond formation

•translocation (GTP)



Termination~ ‘stop’ codon, release factor binds ‘A’ site, bring s H

2

O



Polyribosomes: translation of

mRNA by many ribosomes (many

copies of a polypeptide very quickly)

(16)

Building a Protein: Review

Figure 17.18

Amino end of polypeptide

mRNA Ribosome ready for

next aminoacyl tRNA

E

P A

E

P A

E

P A

E

P A

GDP

GTP

GTP GDP 2

2 site site

5

3

TRANSCRIPTION

TRANSLATION

DNA

mRNA Ribosome Polypeptide

http://www.hhmi.org/biointeractiv http://www.hhmi.org/biointeractiv e/translation-advanced-detail e/translation-advanced-detail

Small

ribosomal su

& tRNA-Met bind, then attach to 5’

cap, scan until start codon / anticodon bind. Large su then

attaches

(17)

Building a Protein: Review



Simple Translation

– http://www.youtube.com/watch?v=KvYEqGb7XN8&NR=1



http://www.johnkyrk.com/er.html



http://www.hhmi.org/biointeractive/dna-transcriptio n-advanced-detail



Transcription & Translation:

– http://www.youtube.com/watch?v=D3fOXt4MrOM&f eature=related



Bio Rap



http://www.youtube.com/watch?v=d1UPf7lXeO8

(18)

Polyribosome

Figure 17.20a, b

Growing polypeptides

Completed polypeptide

Incoming ribosomal subunits

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)

(19)

Some Ribosomes Remain Free in the Cytoplasm While others attach to ER



A Signal Recognition Particle(srp) attaches to a signal peptide



The srp binds a receptor protein on the ER



These proteins will become part of the nuclear envelope, ER, golgi, lysosomes, vacuoles or secretory proteins

Figure 17.21

Ribosome

mRNA Signal peptide Signal-

recognition particle (SRP) SRP

receptor protein

Translocation complex CYTOSOL

Signal peptide removed

ER membrane

Protein

ERLUMEN

https://www.youtube.com/watch?v=4qf1BSXn_tk

htt ps:

//w ww.

you tub e.c om/

wat ch?

v=X

CLf

dGb

ySA

A

(20)

Mutations: genetic material changes in a cell

 POINT MUTATIONS….

 Changes in 1 or a few base pairs in a single gene

 Base-pair substitutions: •silent mutations no effect on protein

•missense

∆ to a different amino acid (different protein) •nonsense ∆ to a stop

codon and a nonfunctional protein

 Base-pair insertions or deletions: additions or losses of nucleotide pairs in a gene; alters the ‘reading frame

’ of triplets~frameshift mutation

 Mutagens: physical and

chemical agents that change DNA

(21)

Sickle Cell Anemia - AA Substitution

Primary structure Secondary and tertiary structures

Quaternary structure

Function

Red blood cell shape

Hemoglobin A

Molecules do not associate with one another, each carries oxygen.

Normal cells are full of individual hemoglobin molecules, each carrying oxygen







10 m 10 m





  Primary

structure Secondary and tertiary structures

Quaternary structure Function

Red blood cell shape

Hemoglobin S Molecules interact with one another to crystallize into a fiber, capacity to carry oxygen is greatly reduced.

 subunit  subunit

1 2 3 4 5 6 7 1 2 3 4 5 6 7

Normal hemoglobin Sickle-cell hemoglobin

. . . . . .

Figure 5.21

Exposed hydrophobic

region

Val His Leu Thr Pro Glul Glu Val His Leu Thr Pro Val Glu

(22)

RNA plays multiple roles

(23)

Types of RNA

http://www.biology-pages.info/T/Transcription.html



◦Messenger RNA(mRNA)



◦Ribosomal RNA (rRNA)



◦Transfer RNA (tRNA)



◦Small Nuclear RNA (snRNA)



◦Small Nucleolar RNA(snoRNA)



◦MicroRNA (miRNA)



◦ XIST RNA (an example of Long noncoding or

Lnc RNA)

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