Formation of RNA Polymerase II pre-initiation complex

IIA stabilizes IID binding to promoter

IID contains TBP that binds TATA box

IIB binds initiation sequence

IIE stimulates transcription

IIH has kinase and helicase activity

Pol II binds IIB

RNA Synthesis in E. Coli

Transcription bubble

RNA splicing in eukaryotes

Primary transcript,hnRNA

Alternative splicing patterns give rise to multiple proteins from the same pre-mRNA

RNA Synthesis: Take Home Message

1) DNA sequences are translated into RNA messages by RNA polymerases.

2) The initiation of RNA synthesis is controlled by specific DNA promoter sequences.

3) The synthesis of RNA is governed by initiation, elongation, and termination steps.

4) Eukaryotic mRNA is extensively processed

Overview of Protein Synthesis (Translation). Transfer RNA.

 

Required reading: Stryer’ Biochemistry 5th edition Ch. 5, p. 132-136,

Ch. 28 p. 797, Ch. 29 p. 813-823or Stryer 4th edition p. 102-104, 109-112, Ch. 34, p. 875-888 and

Ch. 33 p. 849-850

Flow of Genetic Information

DNA RNA Proteins Cellular Action

transcription translation

DNA

rep

licat

ion

How do we go from mRNA to Protein?

DNA mRNA Protein

t-RNA

Amino Acid Sequence

O

N

NN

N

NH2

O

O

HH

HH

PO

O

tRNA

CO

CH

R

NH3

OH

o-

Transfer RNA

• Acts as an adaptor molecule between mRNA and peptide sequence• Contains amino acid attachment site and template recognition site

aminoacyl tRNA synthetase

Translation of mRNA

• mRNA is read sequentially from a fixed starting point

• Sequence of 3 Bases = One Codon (no gaps)

• One Codon = One Amino Acid

• 20 Natural Amino Acids = 64 Codons (43)

“Degenerate Code: Several Different Codons per Amino Acid”

5’

N

3’

C

Genetic Code

During translation, mRNA passes through the ribosome so that each

codon recognizes its tRNA

Translating the Message

DNA 5'-ATG-GCC-TTT-GAT-TCT-AAA-TAA-3'

RNA 5'-AUG-GCC-UUU-GAU-UCU-AAA-UAA-3'

Protein N- met ala phe asp ser lys stop -C

Correct reading frame is essential

Write the sequence of amino acids in a polypeptide translated from the following mRNA:

5’ GGA GGA GUA AGU UGUGly– Gly – Val – Ser - Cys

The genetic code is nearly universal, with the exception of mitochondria

Transfer RNA

Secondary Structure of Transfer RNA molecule

H2C NH

NH2C

O

O

dihydrouridine (UH2)

NHHN O

O

pseudouridine (

60-93 nt long

7 bp acceptor stem

Base sequence of yeast tRNAAla: cloverleaf folding result from the presence of “self complementary” regions

-UCCGGTCGAUUCCGGA-

tRNA has an “L” Tertiary Structure

Anti-Codon

Acceptor Stem

V Loop

TLoop

DLoop

Amino acid attachment

site

Tertiary base pairs are responsible for the tertiary structure of tRNA

#46(m7G)

#22G #13

C

Tertiary basepairs in tRNAPhe

#46(m7G)

#22G

#13C

Tertiary basepairs in tRNAPhe

Non-standard H bond interactions, some linking 3 bases, help stabilize the L-shaped tertiary structure of tRNA.

tRNA Formation in E. Coli

CCACCA3'

5'

RNase PRNase P

RNase DRNase D

• 60 genes for tRNA are clustered in 25 transcription units

• tRNA Precursors containing several tRNAs are cleaved with RNases:

RNase P – generates 5’ ends of tRNA by cleaving the bond 5’ to each tRNA

RNase D – trims the 3’ end up to the CCA sequence

tRNA Activation (charging) by aminoacyl tRNA synthetases

AminoacyltRNA synthetase

Two important functions:

1. Implement genetic code

2. Activate amino acids forpeptide bond formation

The key enzymes:Amanoacyl-tRNA synthetases

Aminoacyl-tRNA Synthesis

Summary of 2-step reaction:

1. amino acid + ATP aminoacyl-AMP + PPi

2. aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP

The 2-step reaction is spontaneous overall, because concentration of PPi is kept low by its hydrolysis, catalyzed by Pyrophosphatase.

tRNA Activation by aminoacyl tRNA synthetases

C+H3N

R

O

O(-) PO

O

OH OH

O O-

AdenineO

PO P

O(-)

HO O

O

(-)O

C+H3N

R

O

OP

O

O

OH OH

O O-

Aminoacyl adenylate (Aminoacyl-AMP)

+ PPiAdenine

1. Aminoacyl-AMP formation:

2. Aminoacyl transfer to the appropriate tRNA:

C+H3N

R

O

OAMP+ACC-tRNAC+H3N

R

O

OP

O

O

OH OH

O O-

HO-ACC-tRNAAdenine +

2Pi

Overall reaction: amino acid + tRNA + ATP aminoacyl-tRNA + AMP + PPi

Classes of Aminoacyl-tRNA Synthetases

• Class I: Arg, Cys, Gln, Glu, Ile, Leu, Met, Trp, Tyr, Val (Generally the Larger Amino Acids)

• Class II: Ala, Asn, Asp, Gly, His , Lys, Phe, Ser, Pro, Thr(Generally the smaller amino acids)

Main Differences between the two classes:

1. Structural differences. Class I are mostly monomeric, class II are dimeric.

2. Bind to different faces of the tRNA molecule

3. While class I acylate the 2’ hydroxyl of the terminal Ado,class II synthetases acylate the 3’-OH

Class I and II synthetases bind to different faces of the tRNA molecule

O

N

NN

N

NH2

O

O

HH

HH

PO

O

tRNA

CO

CH

R

NH3

OH

o-

Class I synthetasesacylate the 2’-OH

O

N

NN

N

NH2

O

OH

HH

HH

PO

O

tRNA

CO

CH

R

NH3

O

o-

Class II synthetasesacylate the 3’-OH

The accuracy of protein synthesis depends on correctcharging of tRNAs with amino acids

1. tRNA synthetases must link tRNAs with their correct aminoacids.

2. tRNA synthetases recognize correct amino acids by specificbinding to the active site and proofreading.

3. tRNA synthetases recognize correct tRNAs via by interacting withspecific regions of tRNA sequence.

The accuracy of protein synthesis depends on correctcharging of tRNAs with amino acids

1. tRNA synthetases must link tRNAs with their correct aminoacids.

2. tRNA synthetases recognize correct amino acids by specificbinding to the active site and proofreading.

3. tRNA synthetases recognize correct tRNAs via by specific regions of tRNA sequence.

The acylation site of threonyl tRNA synthetase contains a Zinc ion

that interacts with the OH group of Threonine

H2N CH C

CH

OH

O

OH

CH3

H2N CH C

CH

OH

O

CH3

CH3

Thr Val

Threonyl-tRNA synthetase contains editing site

H2N CH C

CH

OH

O

OH

CH3

Thr

H2N CH C

H2C

OH

O

OH

Ser

tRNA Synthetase Proofreading

•“Double sieve” based on size• Flexibility of the acceptor stem essential

tRNA Synthetase Proofreading

CH3

O+H3N

tRNAIleO

CH3

Smaller Hydrolytic Site

Larger Acylation Site

CH3H3C

ONH3+

O

Larger Acylation Site

Smaller Hydrolytic Site

tRNAIle

CH3

O+H3N

tRNAIleO

CH3

Correct Acylation

H3C CH3

O+H3N

tRNAIleO

Misacylation

Difference in Size

Ile Val

tRNAVal Synthetase Proofreading:

hydrophobic/polar recognition motif

3HC CH3

O+H3N

tRNAValO

Hydrophobic Acylation Site

Polar Hydrolytic Site

CH3 CH3

O+H3N

tRNAValO

Correct Acylation

HO CH3

O+H3N

tRNAValO

Misacylation

OHH3C

ONH3+

OtRNAVal

Polar Hydrolytic Site

Hydrophobic Acylation Site

Difference in Hydrophobicity

Val Thr

The accuracy of protein synthesis depends on correctcharging of tRNAs with amino acids

1. tRNA synthetases must link tRNAs with their correct aminoacids.

2. tRNA synthetases recognize correct amino acids by specificbinding to the active site and proofreading.

3. tRNA synthetases recognize correct tRNAs via using specific regions of the tRNA sequence.

tRNA Recognition by Synthetases

• different recognition motif depending on synthetase

• usually just a few bases are involved in recognition

•Can involve specific recognition of the anticodon (e.g. tRNAMet), stem sequences can (e.g. tRNAAla), both stem regions and anticodon (e.g. tRNAGln), or, less frequently, D loop or T loop bases.

A

OH

P5'

3'

U70G3

A C C

OH

P5'

3'A C C

OH

P5'

3'A C C

tRNAAla tRNAPhe

G34A35

A36

Examples of tRNA Recognition by aminoacyl tRNA Synthetases

tRNASer

C11

G24

D

Threonyl tRNA synthase complex with tRNA

Codon-anticodon recognition between tRNA and mRNA

The relationship between the number of codons, tRNAs, and synthetases

Total of 61 codons, but not 61 tRNAs!

The same tRNA can recognize more than one codon

Example:

Codon tRNA Synthetase

GCUGCC tRNAAla (5’-IGC-3’) alanyl tRNA synthetaseGCA

CGI anticodon

5’-GCU (C,A)-3’ codon

5’3’

Codon : Anticodon Recognition

t RNA- 3'-X Y Z -5' anticodonmRNA- 5'-X’Y’Z’-3' codon

1 2 3

3 2 1

The Third Base of Codon is Variable

1. The first two interactions (XY-X’Y’) obey Watson-Crickbase pairing rules.

2. The third interaction is less strict (Wobble pairing is allowed)

Wobble base pairing rules

5’ anticodon base 3’ codon base

C G

A U

U A or G

G C or U

I U, C, or A

tRNA Anticodon-Codon Recognition

HN

N N

N

O

Inosine

Ribose

N

N N H

N

NH2

HN

HN

N N H

N

O

Adenosine Guanosine

C G IG C C

C G IG C A

C G IG C UCodon

Anticodon 5'3'3'5'

5'3'3'5'5' 3'

3' 5'

NHN

N N

O

C1'

N

N

NH2

OC1'

C-I base pair

NN

NHN

NH2

NHN

N N

O

C1'A-I base pair

C1'

NH

N

O

O

N

HN

NN

O

C1'C1'

U-I base pair

tRNA Anticodon-Codon Recognition

C G IG C U

C G IG C C

C G IG C ACodon

Anticodon 5'3'3'5'

5'3'3'5'5' 3'

3' 5'

C G GG C U

C G GG C CCodon

Anticodon 5'3'3'5'5' 3'

3' 5'

C G UG C A

C G UG C GCodon

Anticodon 5'3'3'5'5' 3'

3' 5'

C G CG C G

C G AG C UCodon

Anticodon 5'3'3'5'5' 3'

3' 5'

Genetic Code

Nonsense suppression

Nonsense mutations = change of codon for an aa to STOP

Usually lethal – truncated protein

Can be rescued by mutation in a different part of the genome

Mechanism: tRNA gene mutation

Example: E. Coli Amber suppressortRNATyr anticodon change GUA CUAMutated tRNA recognized stop codon as Tyr and prevents chain termination

Overview of Protein Synthesis : Take Home Message

1) Translation of the genetic code is dependent on three base words that correspond to a single amino acid.

2) The mRNA message is read by tRNA through the use of a three base complement to the three base word.

3) A specific amino acid is conjugated to a specific tRNA (three base word).

4) Amino acid side chain size, hydrophobicity and polarity govern the ability of tRNA synthetases to conjugate a specific three base message with a specific amino acid.