From Gene to Protein

Chapter 17

Overview: The Flow of Genetic Information

• the information content of DNA is in the form of specific sequences of nucleotides

• the DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins

• proteins are the links between genotype and phenotype• Gene expression = process by which DNA directs protein synthesis– includes two stages: transcription and translation

Basic Principles of Transcription and Translation

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

• Transcription = synthesis of RNA – using information in DNA

• Translation = synthesis of a polypeptide– using information in the mRNA– Ribosomes - sites of translation

DNA RNA Protein

The Products of Gene Expression: A Developing Story

• original hypothesis posed by scientists: one gene – one enzyme

• BUT a lot of proteins aren’t enzymes - researchers later revised the hypothesis: one gene–one protein

• many proteins are composed of several polypeptides– each of which has its own gene

• can now restated the hypothesis as the one gene–one polypeptide hypothesis

• **Note: common to refer to gene products as proteins rather than polypeptides

DNA vs. RNA

Types of RNA mRNA = messenger RNA

– majority of RNA found in a cell– carries the genetic information which will be translated into a

protein sequence– defined by the presence of a “cap” at its 5’ end and a long tail of

adenines at its 3’ end = “poly-A tail”

Types of RNA

rRNA = ribosomal RNA found in the nucleolus combines together with the large and small ribosomal subunits to

form the functional ribosome (protein translation) rRNA is transcribed in the nucleolus by RNA polymerase I

28S rRNA

Types of RNA

tRNA = transfer RNA actually translates the message coded in the mRNA into a protein

sequence which will become a function protein tRNA is transcribed in the nucleoplasm by an enzyme called RNA

polymerase III then exported into the cytoplasm where AA are added

-transcription of RNA is similar to DNA replication – RNA is made in the 5’ to 3’ direction-enzyme called an RNA polymerase binds to only one of the DNA strands = the anti-sense (template strand)-it moves along the template DNA strand (in the 3’ to 5’ direction) and reads the nucleotide and adds a complementary RNA base - a growing strand of RNA complementary to the DNA strand results-BUT rather than a T being paired with an A – U becomes the partner to A

5’ 3’

3’ 5’

Transcription

-a human gene is also known as a transcription unit = stretch of DNA that is transcribed into RNA

-a transcription units is comprised of:1. coding sequence – gives rise to protein strand upon translation

-contains regions of code = “exons” – code for amino acids-and regions of junk = “introns” – spliced out in the nucleus

Intron Intron IntronExon ExonExonExon5’3’

Transcription

- 2. untranslated regions (UTRs) - the regions upstream and downstream of the coding region that are transcribed but NOT translated into a protein- -play an important role in translation – can influence the binding of the ribosome

to the mRNA- -also play a role in exporting the mRNA into the cytoplasm

Transcription

• genes are also associated with additional sequences of DNA1. core promoter sequence – for the binding of the RNA polymerase

-RNA polymerase recognizes specific sequences of nt’s-binding is helped out by transcription factors

2. enhancer regions – help enhance transcription can be several thousands of base pairs upstream of the gene

Transcription• the transcription unit is transcribed by an RNA polymerase • three types of RNA polymerase – I, II and III• RNA polymerases create an RNA strand called a primary transcript

• must be modified to produce the final mRNA, tRNA or rRNA• RNA polymerase II transcribes protein coding genes into a primary transcript called pre-

mRNA – this is then is processed into mRNA– genes for tRNA are transcribed in the cytoplasm by RNA polymerase III – primary

transcript is modified into tRNA– genes for rRNA is transcribed in the nucleolus by RNA polymerase I – primary

transcript is modified into rRNA

-3D representation of theRNA polymerase II enzyme

Transcription• three stages of transcription

– Initiation: binding of the RNA polymerase to the promoter• special sequences denote this region

– Elongation: movement of the RNA polymerase along the anti-sense DNA strand and synthesis of the RNA transcript

– Termination: release of the RNA polymerase from the DNA• special sequences denote this region• differs between prokaryotes and eukaryotes

Promoter

RNA polymeraseStart point

DNA

53

Transcription unit

35

1. Initiation – RNA polymerase binds to a special sequenceof nucleotides called the promoter

-certain sections of the promoter are important in polymerase binding = core promoter-in prokaryotes the promoter binds the RNA polymerase without help-in eukaryotes – the polymerase requires the assistance of proteins called transcription factors-specific transcription factors bind to the promoter first and then help position the polymerase at the promoter-additional transcription factors then bind-entire complex is called the Transcription Initiation Complex

Transcription initiationcomplex forms

3

DNAPromoter

Nontemplate strand

53

53

53

Transcriptionfactors

RNA polymerase IITranscription factors

53

53

53

RNA transcript

Transcription initiation complex

5 3

TATA box

T

T T T T T

A A A A A

A A

T

Several transcriptionfactors bind to DNA

2

A eukaryotic promoter1

Start point Template strand

sequence given in texts is that of the sense strand

Promoter

RNA polymeraseStart point

DNA

53

Transcription unit

35

Initiation

53

35

Nontemplate strand of DNA

Template strand of DNARNAtranscriptUnwound

DNA

1

1. Initiation cont… -RNA polymerase unwinds the DNA helix (acts as a helicase) – exposes about 10 to 20 nucleotides for copying -RNA polymerase holds the DNA helix open (acts like the SSBs)-RNA polymerase initiates RNA synthesis without the need for a primer

Promoter

RNA polymeraseStart point

DNA

53

Transcription unit

35

Elongation

53

35

Nontemplate strand of DNA

Template strand of DNARNAtranscriptUnwound

DNA2

3535

3

RewoundDNA

RNAtranscript

5

Initiation1

2. Elongation – RNA polymerase synthesizes a complementary RNA strand-RNA primary transcript grows in the 5’ to 3’ direction-uses uracil instead of thymine-the DNA strands reform their helix once the RNA polymerase moves past the area-the mRNA strand emerges from the polymerase-DNA complex

Nontemplatestrand of DNA

RNA nucleotides

RNApolymerase

Templatestrand of DNA

3

35

5

5

3

Newly madeRNA

Direction of transcription

A

A A A

AA

A

T

TT

T

TTT G

GG

C

C C

CC

G

C CC A AA

U

U

U

end

Multiple RNA polymerases per DNA template

Promoter

RNA polymeraseStart point

DNA

53

Transcription unit

35

Elongation

53

35

Nontemplate strand of DNA

Template strand of DNARNAtranscriptUnwound

DNA2

3535

3

RewoundDNA

RNAtranscript

5

Termination3

35

5Completed RNA transcript

Direction of transcription (“downstream”)

53

3

Initiation1

3. Termination – RNA polymerase reaches a specific sequence of nucleotides and stops transcription-the RNA polymerase detaches from the DNA-the pre-RNA primary transcript is released

-in prokaryotes – a termination sequence that detaches the polymerase-in eukaryotes – the RNA polymerase transcribes a sequence called a poly-adenylation signal– for the release of the pre-RNA from the polymerase

Transcription• to modify the primary transcript into mRNA – the

following modifications are made:– a 5’methylated cap is added to the 5’end– addition of a 3’ poly A tail– the coding sequence is “edited” = splicing

Eukaryotic cells modify RNA after transcription

• enzymes in the eukaryotic nucleus modify pre-mRNA before exporting the mRNA to the cytoplasm– known as RNA processing

• 5’ methylated cap – plays a role in the docking of the ribosome to mRNA – for translation– modified guanine nucleotide added after the transcription of about 20 to 40

nucleotides

Protein-codingsegment

Polyadenylationsignal

5 3

35 5Cap UTRStartcodon

G P P P

Stopcodon

UTR

AAUAAA

Poly-A tail

AAA AAA…

Eukaryotic cells modify RNA after transcription

• 3’ poly A tail – plays a role in the export of the mRNA into the cytoplasm– after transcription – an enzyme adds 20 to 250 adenine nucleotides after

the poly-adenylation signal sequence– also prevents degradation of the mRNA once its in the cytoplasm

Protein-codingsegment

Polyadenylationsignal

5 3

35 5Cap UTRStartcodon

G P P P

Stopcodon

UTR

AAUAAA

Poly-A tail

AAA AAA…

RNA Splicing• most eukaryotic genes and pre-RNA transcripts have long noncoding stretches of

nucleotides that lie between coding regions– the noncoding regions are called intervening sequences, or introns– coding regions are called exons because they are eventually expressed in the form of a

protein– RNA splicing removes introns and joins exons, creating an mRNA molecule

with a continuous coding sequence– the way you splice can also create multiple isoforms from one RNA transcript

5 Exon Intron Exon

5CapPre-mRNACodonnumbers

130 31104

mRNA 5Cap

5

Intron Exon

3 UTR

Introns cut out andexons spliced together

3

105 146

Poly-A tail

Codingsegment

Poly-A tail

UTR1146

• RNA splicing is carried out by spliceosomes• Spliceosomes = several proteins and small nuclear

ribonucleoproteins (snRNPs) that recognize specific sequences found in introns called splice sites

• snRNPs – found in the nucleus and are made of small nuclear RNA (snRNA) and proteins

RNA transcript (pre-mRNA)5

Exon 1

Protein

snRNA

snRNPs

Intron Exon 2

Other proteins

RNA transcript (pre-mRNA)5

Exon 1

Protein

snRNA

snRNPs

Intron Exon 2

Other proteins

Spliceosome

5

Spliceosomecomponents

Cut-outintronmRNA

5Exon 1 Exon 2

1. snRNPs and other proteinscombine to form the spliceosome

2. the spliceosome brings the endsof two exons together -forms a “lariat” out of the intron

3. the spliceosome cuts thepre-mRNA and releases the intronfor degradation

RNA Splicing• genes can encode for more than one

protein– depending on what segments of RNA are

treated as exons and what are treated as introns during splicing

• so the way you splice can determine what proteins eventually get made = alternative RNA splicing

• proteins often are composed of discrete regions called domains – coded for by distinct exons– cut out a domain – get a different protein

• also - exon shuffling may result in the evolution of new proteins– introns increase the probability of crossing-

over between alleles– creates new exon combinations

Gene

DNA

Exon 1 Exon 2 Exon 3Intron Intron

Transcription

RNA processing

Translation

Domain 3

Domain 2

Domain 1

Polypeptide

Splicing• for an animation go to

http://sumanasinc.com/webcontent/animations/content/mRNAsplicing.html

• (don’t worry about the actual proteins!)

Translation• process of converting an mRNA message into a strand of amino acids that will be

processed into a mature functional protein• performed by the ribosome in combination with tRNA molecules• prokaryotes - translation of mRNA can begin before transcription has finished – no

separation between the mRNA and the ribosome• eukaryotic cell- the nuclear envelope separates transcription from translation

– mRNA has to be exported out of the nucleus first

DNAtemplatestrand

TRANSCRIPTION

mRNA

TRANSLATION

Protein

Amino acid

Codon

Trp Phe Gly

5

5

Ser

U U U U U3

3

53

G

G

G G C C

T

C

A

A

AAAAA

T T T T

T

G

G G G

C C C G GDNAmolecule

Gene 1

Gene 2

Gene 3

C C

• How are the instructions for assembling amino acids into proteins encoded into DNA?

• 20 amino acids - only four nucleotide bases in DNA

• how many nucleotides correspond to an amino acid?

• the mRNA nucleotide sequence is “read” in groups of 3 nucleotides = “codons”

• each codon codes for 1 of the 20 amino acids that make up proteins

• called the “genetic code”– 61 amino acid codons; 3 stop codons

• the code is redundant - each amino acid can be coded for by more than one codon

• e.g. alanine – GCU, GCC, GCA and GCG• the GC defines the amino acid as alanine

• in many cases the 3rd codon is important in defining the amino acid– serine –codons are: AGU, AGC– BUT arginine codons are: AGA and AGG

The Genetic Code

Second mRNA base

Fir

st m

RN

A b

ase

(5

end

of

cod

on

)

Th

ird

mR

NA

bas

e (3

en

d o

f co

do

n)

UUU

UUC

UUA

CUU

CUC

CUA

CUG

Phe

Leu

Leu

Ile

UCU

UCC

UCA

UCG

Ser

CCU

CCC

CCA

CCG

UAU

UACTyr

Pro

Thr

UAA Stop

UAG Stop

UGA Stop

UGU

UGCCys

UGG Trp

GC

U

U

C

A

U

U

C

C

CA

U

A

A

A

G

G

His

Gln

Asn

Lys

Asp

CAU CGU

CAC

CAA

CAG

CGC

CGA

CGG

G

AUU

AUC

AUA

ACU

ACC

ACA

AAU

AAC

AAA

AGU

AGC

AGA

Arg

Ser

Arg

Gly

ACGAUG AAG AGG

GUU

GUC

GUA

GUG

GCU

GCC

GCA

GCG

GAU

GAC

GAA

GAG

Val Ala

GGU

GGC

GGA

GGGGlu

Gly

G

U

C

A

Met orstart

UUG

G

1964

Molecular Components of Translation

• two components• 1. transfer RNA (tRNA)• 2. the ribosome

Amino acidattachmentsite

3

5

Hydrogenbonds

Anticodon

(a) Two-dimensional structure (b) Three-dimensional structure(c) Symbol used

in this book

Anticodon Anticodon3 5

Hydrogenbonds

Amino acidattachmentsite5

3

A A G

• tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long

• at one end – anticodon site for the hybridization with the mRNA template• at the other end – attachment site for the amino acid that corresponds to the

mRNA codon• transcribed in the cytoplasm by RNA polymerase III – it folds into its

characteristic shape spontaneously due to regions that complement each other

tRNA

Aminoacyl-tRNAsynthetase (enzyme)

Amino acid

P P P Adenosine

ATP

P

P

P

PPi

i

i

Adenosine

tRNA

AdenosineP

tRNA

AMP

Computer model

Aminoacid

Aminoacyl-tRNAsynthetase

Aminoacyl tRNA(“charged tRNA”)

-amino acids are attached inthe cytoplasm by enzymes called aminoacyl-tRNA –synthetases-one end fits the amino acid,the other end fits the tRNA-20 synthetases – each is specificfor only one kind of tRNA-the tRNA attached to an AA iscalled a ‘charged tRNA’

tRNA and the 3rd codon “wobble”

• the tRNA recognizes the codon “triplet” on the mRNA template

• attached to the tRNA is the amino acid corresponding to this codon

• there are 61 amino acid codons – so there should be 61 tRNAs

• there are only 45 tRNAs– some tRNAs can bind more than one codon

• the rules for complementary base pairing at the third NT of the codon are less stringent– “flexible” base pairing at this NT = Third Codon Wobble

Ribosomes• machine of translation• made in the nucleolus in eukaryotic cells• comprised of two subunits of proteins (large and small) linked

together with a piece of rRNA– eukaryotes: 40S small subunit = 33 proteins + 18S rRNA

+ 60S large subunit = 50 proteins + 28S rRNA (+ 5.6S rRNA + 5S rRNA)– rRNA is transcribed in the nucleolus, proteins are imported from cytoplasm – everything is assembled in the nucleolus– subunits are exported out via nuclear pores– prokaryotic ribosomes and similar but smaller

Ribosomes• within the large subunit are two sites for the binding of tRNA

– P-site or Peptidyl-tRNA site – “old” AA– A-site or aminoacyl-tRNA site – incoming AA

• and one E site/Exit site for the exit of the tRNA off the ribosome

Exit tunnel

A site (Aminoacyl-tRNA binding site)

Smallsubunit

Largesubunit

P A

P site (Peptidyl-tRNAbinding site)

mRNAbinding site

E site (Exit site)

E

Ribosomes• eukaryotic ribosomes are similar but are larger vs. prokaryotes• most evidence now identifies the rRNA as being the catalyst for

the formation of the peptide bond and the growth of the polypeptide chain– RNA with enzymatic activity = ribozyme

Amino end

mRNA

E

(c) Schematic model with mRNA and tRNA

5 Codons

3

tRNA

Growing polypeptide

Next aminoacid to beadded topolypeptidechain

Building a Polypeptide• 3 stages of translation:

– Initiation– Elongation– Termination

• all three stages require protein “factors” – called initiation factors or IFs– in eukaryotes – known as eIFs

• the small subunit of the ribosome binds onto the mRNA sequence near the 5’ methylated cap • this subunit already has an initiator tRNA (bound to methionine) associated with it

• binding of the small subunit is helped by numerous eukaryotic initiation factors (eIFs)• the small subunit then glides down the mRNA “scanning” for the first codon - START codon

= AUG (methionine)-stops so that initiator tRNA can hybridize with the start codon

1. Initiation of Translation

InitiatortRNA

mRNA

5

53Start codon

Smallribosomalsubunit

mRNA binding site

3

Translation initiation complex

5 33 U

UA

A GC

P

P site

i

GTP GDP

Met Met

Largeribosomalsubunit

E A

5

• once the small subunit is positioned - the large subunit then assembles and completes the ribosomal “machine” • helped by even more eIF’s• the mRNA and the ribsosome form the Translation Initiation

Complex• the eIF’s are released once this complex forms• the ribosome is now ready for the next AA - elongation

follows

InitiatortRNA

mRNA

5

53Start codon

Smallribosomalsubunit

mRNA binding site

3

Translation initiation complex

5 33 U

UA

A GC

P

P site

i

GTP GDP

Met Met

Largeribosomalsubunit

E A

5

2. Elongation of Translation

http://www.youtube.com/watch?v=5bLEDd-PSTQhttp://www.youtube.com/watch?v=Ikq9AcBcohAhttp://www.youtube.com/watch?v=NJxobgkPEAo

2. Elongation of Translation

3. Termination of Translation

Releasefactor

Stop codon(UAG, UAA, or UGA)

3

5

3

5

Freepolypeptide

5

32 GTP

2 GDP 2 P

-translation also stops at specific codons = STOP codons-UAA, UGA, UAG

-so when the ribosome reaches these sequences – no more AAs are added and the ribosome detaches from the peptide strand and mRNA-a release factor cleaves the polypeptide chain from the tRNA and releases it from the ribosome (GTP hydrolysis)-the translation machine “breaks apart” – requires an enzyme that uses ATP hydrolysis

Polyribosomes• a number of ribosomes can

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

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

Completedpolypeptide

Incomingribosomalsubunits

Start ofmRNA(5 end)

End ofmRNA(3 end)

(a)

Polyribosome

Ribosomes

mRNA

(b)0.1 m

Growingpolypeptides