©2001 timothy g. standish proverbs 3:1-3 1my son, forget not my law; but let thine heart keep my...

©2001 Timothy G. Standish

Proverbs 3:1-3

1 My Son, forget not my law; but let thine heart keep my commandments:

2 For length of days, and long life, and peace, shall they add to thee.

3 Let not mercy and truth forsake thee: bind them about thy neck; write them upon the table of thine heart:

©2001 Timothy G. Standish

MessengerMessengerRNARNA

Timothy G. Standish, Ph. D.

mRNA

Transcription

IntroductionIntroduction

The Central Dogma The Central Dogma of Molecular Biologyof Molecular Biology

Cell

Polypeptide(protein)

TranslationRibosome

Reverse tanscription DNA

©2001 Timothy G. Standish

Information Only Goes One WayInformation Only Goes One WayThe central dogma states that once “information” has

passed into protein it cannot get out again. The transfer of information from nucleic acid to nucleic acid, or from nucleic acid to protein, may be possible, but transfer from protein to protein, or from protein to nucleic acid, is impossible. Information means here the precise determination of sequence, either of bases in the nucleic acid or of amino acid residues in the protein.

Francis Crick, 1958

©2001 Timothy G. Standish

TranscriptionTranscription

5’

3’

3’

5’

Template (antisense) strand

Coding (sense) strand

©2001 Timothy G. Standish

TranscriptionTranscription

5’

3’

3’

5’

Template (antisense) strand

Coding (sense) strand

5’

RNA

RNAPol.

©2001 Timothy G. Standish

TranscriptionTranscription

5’

3’

3’

5’

5’

RNAPol.

Template (antisense) strand

Coding (sense) strand

©2001 Timothy G. Standish

RNA PolymeraseRNA PolymeraseRNA Polymerase is a spectacular

enzyme, it performs the following functions:

Recognition of the promoter regionMelting of DNA (Helicase +

Topisomerase)RNA Priming (Primase)RNA PolymerizationRecognition of terminator sequence

©2001 Timothy G. Standish

Products of TranscriptionProducts of Transcription Transcription produces three major RNA products:

1 Ribosomal RNA (rRNA) - Several rRNAs are vital constituents of ribosomes

2 Transfer RNA (tRNA) - The molecule that physically couples nucleic acid codons with specific amino acids

3 Messenger RNA (mRNA) - The nucleic acid messenger that carries encoded information from genes on DNA to the protein manufacturing ribosomes

©2001 Timothy G. Standish

Stages of TranscriptionStages of Transcription

Transcription can be logically divided into three distinct stages:

1. Initiation

2. Elongation

3. Termination

©2001 Timothy G. Standish

A “Simple” GeneA “Simple” Gene

Protein Coding Region

Terminator Sequence

Promoter/Control Region

Transcription Start Site 3’ Untranslated Region

5’ Untranslated Region

3’5’

RNA Transcript

©2001 Timothy G. Standish

Transcription InitiationTranscription Initiation

Proteins called transcription factors bind to the promoter region of a gene

If the appropriate transcription factors are present, RNA polymerase binds to form an initiation complex

RNA polymerase melts the DNA at the transcription start site

Polymerization of RNA begins

©2001 Timothy G. Standish

RNA Pol.

InitiationInitiation

T. F.

RNA Pol.

5’RNA

Promoter

T. F.

T. F.

©2001 Timothy G. Standish

Transcription TerminationTranscription TerminationThere are two types of termination: Rho dependent requires a protein called

Rho, that binds to and slides along the RNA transcript. The terminator sequence slows down the elongation complex, Rho catches up and knocks it off the DNA

Rho independent termination depends on both slowing down the elongation complex, and an AT rich region that destabilizes the elongation complex

©2001 Timothy G. Standish

RNAPol.

5’RNA

RNA Pol.

5’RNA

RNA Pol.

5’RNA

TerminationTerminationRho IndependentRho Independent

Terminator

©2001 Timothy G. Standish

RNAPol.

5’RNA

TerminationTerminationRho DependentRho Dependent

Terminator

RNA Pol.

5’RNA

RNA Pol.

5’RNA

Help, rhohit me!

©2001 Timothy G. Standish

DifferencesDifferencesBetween Transcription InBetween Transcription In

Prokaryotes and Prokaryotes and EukaryotesEukaryotes

©2001 Timothy G. Standish

3’

5’

5’

3’

Transcription And Translation Transcription And Translation In ProkaryotesIn Prokaryotes

Ribosome

Ribosome5’

mRNA

RNAPol.

©2001 Timothy G. Standish

DNA

Cytoplasm

Nucleus

Eukaryotic TranscriptionEukaryotic Transcription

ExportG AAAAAA

RNA

Transcription

Nuclear pores

G AAAAAA

RNAProcessing

mRNA

©2001 Timothy G. Standish

A “Simple” Eukaryotic GeneA “Simple” Eukaryotic Gene

Terminator Sequence

Promoter/Control Region

Transcription Start Site

RNA Transcript

5’ Untranslated Region 3’ Untranslated Region

Exons

Introns

3’5’ Exon 2 Exon 3Int. 2Exon 1 Int. 1

3’5’ Exon 2 Exon 3Exon 1 Int. 2Int. 1

©2001 Timothy G. Standish

3’5’ Exon 2 Exon 3Int. 2Exon 1 Int. 1

Processing Eukaryotic mRNAProcessing Eukaryotic mRNA

Protein Coding Region

3’ Untranslated Region5’ Untranslated Region

3’AAAAA

3’ Poly A Tail

5’ G

5’ Cap

Exon 2 Exon 3Exon 1

Int. 2

Int. 1

RNA processing achieves three things: Removal of introns Addition of a 5’ cap Addition of a 3’ tail

This signals the mRNA is ready to move out of the nucleus and may control its life span in the cytoplasm

©2001 Timothy G. Standish

The 5’ CapThe 5’ Cap

HH

C

O

C

N

C

O

NC

C

C

C

C

H

HO

H

OP

O

HOH

O

5’ End of hnRNA

C

N

ON C

C

CH

HO

P

O

HO

O

P

OHH

OO

P

OOH

O

GTP

©2001 Timothy G. Standish

The 5’ CapThe 5’ Cap

H H

C

O

C

N

C

O

NC

C

C

C

C

H

H OH

H

O P

O

H

O

P

O

O

HO

O

P

O

HHO

OHHH

C

O

C

N

C

O

NC

C

C

C

C

H

HO H

H

OP

O

HO

O

©2001 Timothy G. Standish

Transfer RNA (tRNA)Transfer RNA (tRNA) Acts as the adapter molecule between the genetic code

on mRNA and the protein “language” 75-85 bases long A specific amino acid is covalently linked at the 3’ end Elsewhere on the molecule is an anticodon

complimentary to the specific amino acid codon on mRNA that codes for the amino acid carried by the tRNA

Contain a number of modified bases

©2001 Timothy G. Standish

D Arm - Contains dihydrouridine

Acceptor Arm - A specific amino acid is attached to the 3’ end

TC arm - stands for pseudouridine

Extra Arm - May vary in size

Anticodon

Transfer RNA (tRNA)Transfer RNA (tRNA)

U*

9

262223Pu

16

12Py 10

25

20:1

G*

17:1

Pu

A20:2

1713

20G

A5051

656463

G

62

52

CPu

59

A*

C

Py

T49

39

4142

31

2928

Pu*

43127

U35

38

36

Py*

34

403047:1

47:15

46

Py47:16

4544

47

73CCA

707172

66676869

321

7654

Amino Acid attachment site

©2001 Timothy G. Standish

Aminoacyl-tRNA SynthetaseAminoacyl-tRNA Synthetase

Aminoacyl-tRNA Synthetase enzymes attach the correct amino acids to the correct tRNA

This is an energy consuming processAminoacyl-tRNA Synthetases recognize

tRNAs on the basis of their looped structure, not by direct recognition of the anticodon

GlyAmino-acyl-tRNASynthetase

GlyAmino-acyl-tRNASynthetase

AP

MakingMakingAminoacyl-Aminoacyl-

tRNAtRNA

PPPyrophosphate

AP

PP

ATP

Amino-acyl-tRNASynthetase

AP

Gly

CCA

Amino-acyl-tRNASynthetase

MakingMakingAminoacyl-Aminoacyl-

tRNAtRNA

Gly

CCA

Aminoacyl-tRNA

Note that the amino acid is not paired with the tRNA on the basis of the anticodon. The correct tRNA for a given amino acid is recognized on the basis of other parts of the molecule. ©1998 Timothy G. Standish

Gly

PPPyrophosphate

AP

PP

ATP

Amino-acyl-tRNASynthetase

GlyAmino-acyl-tRNASynthetase

AP

APAMP

Amino-acyl-tRNASynthetase

Gly

CCA

©2001 Timothy G. Standish

AminoacylationAminoacylationof tRNAof tRNA

H

H

C

O

C

N

C

O

N

C

C

C

C

CH

HO

H

HO

P

OH

O

N

N

C

C

O

H H

H

N

H

H

3’3’5’5’

H CN

C

O

H

R H

HO

HO H

©2001 Timothy G. Standish

Aminoacid

tRNA

AminoacylationAminoacylationof tRNAof tRNA

3’3’5’5’

H

H

C

O

C

N

C

O

N

C

C

C

C

CH

HO

H

HO

P

OH

O

N

N

C

C

O

H

H

N

H

H

H CN

C

O

H

R H

HO H

Class I Aminoacyl tRNA Synthetases

attach amino acids to the 2’ carbon while

Class II attach tothe 3’carbon

©2001 Timothy G. Standish

Classification of Aminoacyl-Classification of Aminoacyl-tRNA SynthetasestRNA Synthetases

Class I - 2’ OH Glu () Gln () Arg () Val () Ile () Leu () Met ( Tyr (

(

Class II - 3’ OH Gly (2

Ala (4

Pro (

Ser (

Thr (

Asp (??

Asn (

His (

Lys (

Aminoacyl-tRNA Synthetases (ARS) may be mono or multimeric.Two types of polypeptide chains are recognized: and .

©2001 Timothy G. Standish

Requirements for TranslationRequirements for Translation Ribosomes - rRNA and Protiens mRNA - Nucleotides tRNA

– The RNA world theory might explain these three components

Aminoacyl-tRNA Synthetase, – A protein, thus a product of translation and cannot be

explained away by the RNA world theory L Amino Acids ATP - For energy This appears to be an irreducibly complex system

©2001 Timothy G. Standish