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Chapter 17. From Gene to Protein. 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

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slide1

Chapter 17

From Gene to Protein

overview the flow of genetic information
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
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

RNA

DNA

Protein

the products of gene expression a developing story
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

types of 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 rna1
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 rna2
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
slide9

5’

3’

3’

5’

-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

transcription
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

3’

5’

Exon

Exon

Exon

Exon

Intron

Intron

Intron

transcription1
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
transcription2
Transcription
  • genes are also associated with additional sequences of DNA

1. corepromoter 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

transcription3
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 the

RNA polymerase II enzyme

transcription4
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
slide15

2

1

3

Promoter

Transcription unit

A eukaryotic promoter

Promoter

5

3

Nontemplate strand

DNA

3

5

DNA

5

3

T

A

A

A

A

A

T

Start point

5

3

A

T

A

T

T

T

T

RNA polymerase

  • Initiation – RNA polymerase binds to a special sequence
  • of nucleotides called the promoter

TATA box

Template strand

Start point

Several transcriptionfactors bind to DNA

Transcriptionfactors

-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

5

3

3

5

Transcription initiationcomplex forms

RNA polymerase II

Transcription factors

5

3

3

5

3

5

RNA transcript

Transcription initiation complex

sequence given in texts is that of the sense strand

slide16

1

Promoter

Transcription unit

5

3

3

5

DNA

Start point

RNA polymerase

Initiation

Nontemplate strand of DNA

3

5

5

3

Template strand of DNA

RNAtranscript

UnwoundDNA

  • 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
slide17

1

2

Promoter

Nontemplatestrand of DNA

Transcription unit

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

5

3

RNA nucleotides

3

5

DNA

Start point

RNApolymerase

RNA polymerase

Initiation

Nontemplate strand of DNA

3

5

C

C

A

A

T

A

5

3

5

T

3

Template strand of DNA

U

RNAtranscript

T

C

UnwoundDNA

end

3

G

T

Elongation

U

A

G

C

A

RewoundDNA

C

C

U

C

A

A

C

A

3

5

3

5

A

3

5

3

5

T

A

G

G

T

T

RNAtranscript

5

Direction of transcription

Templatestrand of DNA

Multiple RNA polymerases per DNA template

Newly madeRNA

slide18

3

1

2

Promoter

Transcription unit

5

3

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

3

5

DNA

Start point

RNA polymerase

Initiation

Nontemplate strand of DNA

3

5

5

3

Template strand of DNA

RNAtranscript

UnwoundDNA

Elongation

RewoundDNA

3

5

3

5

3

5

RNAtranscript

Termination

3

5

5

3

3

5

Completed RNA transcript

Direction of transcription (“downstream”)

transcription5
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
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

G

P

P

P

AAA

AAUAAA

AAA

Startcodon

Stopcodon

Cap

5

UTR

5

Poly-A tail

3

UTR

eukaryotic cells modify rna after transcription1
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

G

P

P

P

AAA

AAUAAA

AAA

Startcodon

Stopcodon

Cap

5

UTR

5

Poly-A tail

3

UTR

rna splicing
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

Exon Intron

Exon

Intron

Exon

5

3

Poly-A tail

Pre-mRNACodonnumbers

Cap

5

130

31104

105 146

Introns cut out andexons spliced together

5

mRNA

Cap

Poly-A tail

1146

UTR

3

UTR

5

Codingsegment

slide23
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

Intron

Exon 2

Protein

Other proteins

snRNA

snRNPs

slide24

RNA transcript (pre-mRNA)

5

Exon 1

Intron

Exon 2

Protein

Other proteins

snRNA

snRNPs

  • snRNPs and other proteins
  • combine to form the spliceosome

Spliceosome

5

2. the spliceosome brings the ends

of two exons together

-forms a “lariat” out of the intron

Spliceosomecomponents

Cut-outintron

3. the spliceosome cuts the

pre-mRNA and releases the intron

for degradation

mRNA

5

Exon 1

Exon 2

rna splicing1
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

Intron

Exon 2

Intron

Exon 3

Exon 1

Transcription

RNA processing

Translation

Domain 3

Domain 2

Domain 1

Polypeptide

splicing
Splicing
  • for an animation go to http://sumanasinc.com/webcontent/animations/content/mRNAsplicing.html
  • (don’t worry about the actual proteins!)
translation
Translation

DNAtemplatestrand

DNA

5

3

molecule

A

A

A

A

A

C

C

C

C

T

G

G

  • 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

T

T

T

T

A

G

G

G

G

C

T

C

Gene 1

3

5

TRANSCRIPTION

Gene 2

U

G

G

U

U

U

G

G

C

U

C

A

5

3

mRNA

Codon

TRANSLATION

Gly

Phe

Trp

Protein

Ser

Gene 3

Amino acid

the genetic code
The Genetic Code

Second mRNA base

A

U

C

G

UUU

UAU

UCU

UGU

U

Phe

Cys

Tyr

1964

UUC

UCC

UAC

UGC

C

U

Ser

UUA

UCA

UGA Stop

A

UAA Stop

Leu

  • 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

Trp

UUG

UCG

UGG

G

UAG Stop

CUU

CCU

U

CAU

CGU

His

CUC

CAC

CGC

C

CCC

C

Leu

Pro

Arg

CUA

CCA

CGA

A

CAA

Gln

CUG

CCG

CGG

G

CAG

First mRNA base (5 end of codon)

Third mRNA base (3 end of codon)

AUU

AAU

ACU

AGU

U

Ser

Asn

C

AUC

Ile

AAC

ACC

AGC

A

Thr

AUA

AAA

ACA

AGA

A

Lys

Arg

Met orstart

AUG

ACG

AGG

AAG

G

GUU

GCU

GAU

GGU

U

Asp

GUC

GCC

C

GGC

GAC

G

Val

Ala

Gly

Gly

GUA

GCA

GGA

A

GAA

Glu

GUG

GCG

GGG

G

GAG

molecular components of translation
Molecular Components of Translation

two components

1. transfer RNA (tRNA)

2. the ribosome

slide30
tRNA

3

  • 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

Amino acidattachmentsite

5

Amino acidattachmentsite

5

3

Hydrogenbonds

Hydrogenbonds

A

A

G

3

5

Anticodon

Anticodon

Anticodon

(c) Symbol used

(a) Two-dimensional structure

in this book

(b) Three-dimensional structure

slide31

Aminoacyl-tRNAsynthetase (enzyme)

Amino acid

P

Adenosine

P

P

P

Adenosine

P

P

i

Aminoacyl-tRNAsynthetase

ATP

P

tRNA

i

P

i

-amino acids are attached in

the cytoplasm by enzymes

called

aminoacyl-tRNA –synthetases

-one end fits the amino acid,

the other end fits the tRNA

-20 synthetases – each is specific

for only one kind of tRNA

-the tRNA attached to an AA is

called a ‘charged tRNA’

tRNA

Aminoacid

P

Adenosine

AMP

Computer model

Aminoacyl tRNA(“charged tRNA”)

trna and the 3 rd codon wobble
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
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
ribosomes1

A site (Aminoacyl-tRNA binding site)

Ribosomes

P site (Peptidyl-tRNAbinding site)

Exit tunnel

  • 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

E site (Exit site)

E

A

P

Largesubunit

mRNAbinding site

Smallsubunit

ribosomes2
Ribosomes

Growing polypeptide

Amino end

Next aminoacid to beadded topolypeptidechain

  • 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

E

tRNA

mRNA

3

Codons

5

(c) Schematic model with mRNA and tRNA

building a polypeptide
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

slide37

1. Initiation of Translation

  • 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

Largeribosomalsubunit

U

3

5

C

A

P site

Met

Met

3

5

A

G

U

P

i

InitiatortRNA

GTP

GDP

E

A

mRNA

5

5

3

3

Start codon

Smallribosomalsubunit

Translation initiation complex

mRNA binding site

slide38

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

Largeribosomalsubunit

U

3

5

C

A

P site

Met

Met

3

5

A

G

U

P

i

InitiatortRNA

GTP

GDP

E

A

mRNA

5

5

3

3

Start codon

Smallribosomalsubunit

Translation initiation complex

mRNA binding site

slide39

2. Elongation of Translation

http://www.youtube.com/watch?v=5bLEDd-PSTQ

http://www.youtube.com/watch?v=Ikq9AcBcohA

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

slide41

P

3. Termination of Translation

Releasefactor

Freepolypeptide

5

3

3

3

GTP

2

5

5

2

GDP

2

Stop codon

(UAG, UAA, or UGA)

-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
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

Growingpolypeptides

Incomingribosomalsubunits

Polyribosome

Start ofmRNA(5 end)

End ofmRNA(3 end)

(a)

Ribosomes

mRNA

(b)

0.1 m

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