1

DNA base pairs

Base pairingAnti parallel strands

2

Base pairing

DNA sequence (5’ to 3’)

Gene sequence

Intergenic sequence

3

DNA Protein

Eukaryotic Information Transfer: Transcription & Translation

****Beadle and Tatum: Gene = polypeptide****

4

RNA serves as the intermediarybetween DNA and proteins

Although RNA and DNA are structurally analogous,Three major differences

DNA RNA

Four bases

A T G C

Double stranded

Deoxyribose sugar backbone

Four bases

A U G C

Single stranded

Ribose sugar backbone

Most DNA is nuclear Most RNA is cytoplasmic

Genes are in the nucleus

Proteins are made in cytoplasm

5

Transcription

The synthesis of RNA by the enzyme RNA polymerase using DNA as the template is called transcription

For each gene, only one of the two strands of DNA is transcribed

mRNA is an exact copy of a gene that is exported to the cytoplasm

6

Transcription involves THREE distinct processes

RNA polymerase catalyzes the synthesis of RNA using the DNA as a template

RNA polymerase is a multi-protein complexIt consists of four proteins in bacteria (E. coli)

1) Transcription Initiation2) Transcription Elongation3) Transcription Termination

A GENE is a defined region of DNAIt has a start, a body a end.

7

Initiation of Transcription

Initiation involves RNA polymerase recognizingand binding to a specific sequence on the DNA

The recognition sequence is called a PROMOTER

The sequences are present in the promoters of most E. coli genesThese sequences are conservedThey are critical for proper functioning of the promoter

----TTGACAT---------------TATAAT----------AT----ATG CCC GGG TTT TAA----AACTGTA---------------ATATTA----------TA----TAC GGG CCC AAA ATT

(-10)(15-17)(-35)

PROMOTER

5’

3’

3’5’

sense

antisense

What do we mean by conserved sequence?

Regions of the DNA (gene or non-gene) or protein that share similar nucleotide sequence

8

Conservation--Homology

The sequence homology between genes is not usually perfectOnce all the genes are aligned, the most common nucleotide atEach position is used to construct a consensus sequence

(15-17 bp)

Consensus sequences of promoters

----TTGACAT---------------TATAAA----------AT----ATG CCC

(-10)

(-35)

(+1)

T C C G T T G G A C A T T G T T A G T C G C G - C T T G G T A T A A T C G G C FD8 C G T G T T G A C T A T T T T A C C T C T G G - - C G G T T A T A A T G G T C LPR T C C G C T T G A C A T C C T G A T T G C C G A C T C C C T A T A A A G C G C RRNX1 A A C G G T T G A C A A C A T G A A G T A A A - C A C G G T A T G A A G T G A T7A3

T C C G T T T G A C A T T X T G A X T C X C G - C T C G G T A T A A T G G G C Majority

9

Homology (molecular biology)

Regions of the DNA OR PROTEIN (gene or non-gene) that share similar nucleotide sequence

Sequence homology is a very important concept

Structural homology (nucleotide sequence) implies functional homology

Conservation of sequence = Conservation of function

Genes with a similar sequence are likely to function in a similar manner(Homologous genes encode for similar proteins, which will have similar functions)

M A R T K Q T A R K S T G G K A P R K Q L A T mouse H3M A R T K Q T A R K S T G G K A P R K Q L A T Dros H3M A R T K Q T A R K S T G V K A P R K Q L A T Tetra H3M A R T K Q T A R K S T G G K A P R K Q L A S Yeast H3

M A R T K Q T A R K S T G G K A P R K Q L A T Consensus

10

Homology (molecular biology)

Regions of the DNA (gene or non-gene) that share similar nucleotide sequence

Sequence homology is a very important concept

Structural homology (nucleotide sequence) implies functional homology

Conservation of sequence = Conservation of function

Genes with a similar sequence are likely to function in a similar manner(Homologous genes encode for similar proteins, which will have similar functions)

Example:Gene in humans, which when mutated, causes cancer. This gene is identified, isolated, cloned and sequenced.Nothing else is known about this gene in humans

Sequence analysis of this gene indicates that it is homologous to a gene in the fly Drosophila. The gene in the fly encodes for a proteins that is required for DNA replicationIt is very likely that the human gene/protein will be involved in replication

11

RNA polymerase

Bacterial RNA polymerase

Core enzyme:

four polypeptide subunits: alpha (a), beta (b), beta' (b'), and omega (w)

Stoichiometry : 2a:1b:1b’:1w

Core RNA polymerase can bind to DNA It catalyzes the synthesis of RNA but it has no specificity.

(15-17 bp)

Consensus sequences of promoters

----TTGACAT---------------TATAAA----------AT----ATG CCC

(-10)

(-35)

(+1)

12

Sigma

Holo Enzyme:

The RNA polymerase holoenzyme contains an additional subunit - sigma (s). The sigma subunit does two things:

It reduces the affinity of the enzyme for non-specific DNA.

It greatly increases the affinity of the enzyme for promoters. Sigma binds the -35 promoter sequence and targets the polymerase to the promoter

Critical step in regulation of transcription of most bacterial genes is the binding of RNA polymerase to the promoter

(15-17 bp)

Consensus sequences of promoters

----TTGACAT---------------TATAAA----------AT----ATG CCC

(-10)

(-35)

(+1)

13

RNA polymerase

RNA polymerase searches for the promoter

RNA promoter binds the promoterand unwinds the DNA

RNA polymerase synthesizes RNA

14

Promoter asymmetry and direction of transcription

RNA chain length ranges from ~70 to 10,000 nucleotides

The orientation of the promoter defines which DNA strand will be transcribed

Promoter sequence is asymmetrical and orients the binding of the polymerase

--TTGACAT---------------TATAAA----------AT--//-ATG CCC GGG TAA--AACTGTA---------------ATATTT----------TA--//-TAC GGG CCC ATTtemplate

15

RNA polymers are synthesized in the 5’ to 3’ direction

mRNA has the same sequence as the non-template strandOnce the polymerase orientation is established only one DNA strand is read

RNA chains are ONLY made in the 5’ to 3’ direction

The template DNA strand is read in opposite direction (3’ to 5’)

16

Promoters can be found in different relative orientations

17

Gene orientations

For each gene, RNA is transcribed from ONLY ONE DNA strand (template strand)

However different genes may use different DNA strands

Over the entire chromosome, different regions of both DNA strands will be Transcribed

Orientation of genes is the direction in which they are transcribed

5’ 3’

3’ 5’

18

Transcription termination

5’ 3’

5’3’5’ 5’3’ 3’

Termination of transcription requires the protein Rho, that associates with the RNA polymerase, and recognizes a sequence in the mRNA, binds this sequence and terminates transcription by pulling the RNA away from the polymerase. This causes the polymerase to first pause and then dissociate from the DNA strand

Upon termination, the RNA is released from the DNA

Most terminators contain a region rich in GC bases followed by polyU tract. This adopts a hairpin structure in the RNA.

19

General transcription factors

TFIIDTFIIBTFIIFTFIIEPolymeraseTFIIH

These factors bind promoters of ALL GENES

TATA Inr

20

Eukaryotic RNA

Prokaryotes have a single RNA polymeraseThis enzyme synthesizes mRNA, tRNA and rRNA

Eukaryotes have three RNA polymerases

RNA PolymeraseI----rRNA

RNA polymeraseII---mRNA

RNA polymeraseIII--tRNA

RNA is synthesized in the nucleus

This is the Primary transcript

It is processed before being transported to the cytoplasm

5’ cap of 7-methylguanosine is added

3’ polyA tail is added: usually about 150-200 nucleotides long

21

Proximal promoter and Distal Enhancers

The enhancer functions to activate genes. There are specific sequences that bind TISSUE SPECIFIC factors. The binding of these factors induces gene activation 100 fold!

Proximal promoter

Distal enhancer

TATA

+

22

InrTATA Gene

Upstream element function

ProximalPromoter

Transcription Activators bind the enhancer sequences and the promoter sequences. They cooperate together to activate transcription.

Distal Enhancer

DNA binding domainEach activator has a different domain that recognizes a different DNA sequence

Activation domainHelps recruit the general transcription factors

23

Gene specificity

GAL1

GAL2

PHO5

PHO8

Gal4

Gal4

PHO4

Galactose in media

GAL1

GAL2

PHO5

PHO8

Phosphate in media

PHO4

24

Properties of Enhancers

Different Enhancers bind different tissue and cell specific transcription activator proteins and this enables gene activation

Enhancers are orientation independent

Enhancers are distance independent

Enhancers can activate heterologous genes

The enhancer acts as a unit that can be moved relative to the promoter

25

Transcriptional activators bind to enhancers

-Aid in recruitment of the general transcription machinery and assembly of the initiation complex

-Alter binding and/or function of other transcription factors

-Alter rate of transcription initiation

- They recruit enzymes that modify DNA and chromosomal proteins

Mechanism of enhancer function

TATA

26

Mechanism of silencer function

Silencers are orientation independent

Silencers are distance independent

Silencers can repress heterologous genes

The silencer acts as a unit that can be moved relative to the promoter

They recruit repressors

They recruit enzymes that modify DNA and chromosomal proteins

Silencers prevent transcription

27

Establishment of Silencing

Ac Ac Ac Ac Ac Ac Ac Ac1

OR

CR

ap1

Abf1

Ac Ac Ac Ac Ac Ac Ac1

2

4

OR

CR

ap1

Abf1

Ac Ac Ac Ac Ac Ac Ac31

2

4

OR

CR

ap1

Abf1

2

4 Ac Ac Ac Ac Ac Ac31

2

4

OR

CR

ap1

Abf1

2

4 Ac

2

43

2

43

2

43

2

43

2

4331

2

4

OR

CR

ap1

Abf1

28

29

Processing

DNA

Primary transcript

AAAA

Splicing

m1Gppp

m1Gppp

30

Splicing

Internal portions of the primary transcript are removedThis is called splicing

1977 Regions of a gene that code for a protein are interrupted byregions called intervening sequences (introns)

This was discovered by comparing the DNA sequence with the mature cytoplasmic mRNA sequence

Gene7700 nt

1 2 3 4 5 6 7

Ovalbumin

Capping, polyASplicing

mRNA1872 nt

31

Splicing

Primary transcripts are a mosaic of exons and introns

Exons are portions of the mRNA that are translated into protein

Introns (intervening sequences) are segments of the primary Transcript that are removed or spliced out. The function of the intron is not known.

Shuffling of exons allows genes to evolve

Alternative splicing-Different related proteins are synthesized

32

Splicing

Short sequences dictate where splicing occurs

Exon2PuPuGUPuPu--------------Py12-14AG

Exon2

Exon2

Exon1

Exon1

Exon1

Exon1 Exon2

Splicing requires a enzyme complex called a spliceosomeConsists of several small RNAs complexed with ~50 proteins

The snRNA basepair with the splice donor and acceptor sites and are important for holding the two Exons together during splicing

Splice donor Splice acceptor

33

Translation

Translation is the production of a polypeptide whose amino acid sequence is derived from the nucleotide sequence of the mRNA

mRNA is a simple linear molecule made of an array of FOUR different nucleotides

Proteins are complex three dimensional structures made of arrays of 20 amino acids

How do simple mRNA molecules specify complex proteins?

34

Genes, RNA, proteins

Genes synthesize RNAs that are converted to proteins

Genes also encode for RNAs that are NOT converted to proteins

Two major classes of non-protein RNA

tRNA = Transfer RNA

rRNA = Ribosomal RNA

35

Adaptor hypothesis

1958Crick analyzed how RNA made proteins

There are 20 AA

The previous models stated that the mRNA would adopt a Structural configuration forming 20 different cavities- one specific cavity for each AA.

Crick discounted this:”……On physical-chemical grounds, the idea does not seem plausible”

He went on ……”A natural hypothesis is that the amino acidis carried to the template (mRNA) by an adaptor.The adaptor fits onto the mRNA…. And in its simplest form the Hypothesis would require 20 adaptors (one for each amino acid).

“What sort of molecule such adaptors might be is anybody’s guessOne possibility more likely than any other -they contain nucleotides”

“A separate enzyme would join each adaptor to each amino acid”

36

Adaptor hypothesis

tRNA molecules act as adaptors that translate the nucleotide sequence into protein sequence

Each tRNA has two functional sites

Each tRNA is covalently linked to one of the 20 amino acids(a tRNA that specifically carries the amino acid proline is called tRNA-pro)

Each tRNA includes a specific loop (ANTI-CODON loop) that is used to read the mRNA

Proline

GGG |||AAACCCGGG

37

tRNA has a cloverleaf structure

Even though RNA is single stranded and linear, the bases will pair with one another. Complementary bases within the tRNA can pair to form double-stranded regions. This leads to the tRNA adopting a secondary structure(primary structure of a tRNA is the linear nucleotide sequence)

A complete description of all of these base-pairing associations is called the tRNA secondary structure.

This structure is represented as a clover leaf

The three dimensional tertiary structure of tRNA is an L-shaped configuration

38

tRNA has a cloverleaf structure

Even though RNA is single stranded and linear, the bases will pair with one another. Complementary bases within the tRNA can pair to form double-stranded regions. This leads to the tRNA adopting a secondary structure(primary structure of a tRNA is the linear nucleotide sequence)

A complete description of all of these base-pairing associations is called the tRNA secondary structure.

This structure is represented as a clover leaf

The three dimensional tertiary structure of tRNA is an L-shaped configuration

39

Charged tRNA

tRNA are synthesized from genes as RNA

A specific amino acid is then covalently attached to the 3’ end of the tRNA by AA-tRNA synthase (the true translators)

20 synthase enzymes for the 20 amino acids

This tRNA is called a charged tRNAPro

40

tRNA genes, tRNA and charged tRNA

tRNA gene1tRNA1UAC anticodon

Gene tRNAAA-tRNAsynthase Charged tRNA

Met-tRNAsynthase

Met-tRNAUAC

tRNA gene2tRNA2AAA anticodon

Phe-tRNAsynthase

Phe-tRNAAAA

tRNA gene3tRNA3UUU anticodon

Lys-tRNAsynthase

Lys-tRNAUUU

mRNA AUG UUU AAA UAA||| ||| |||

tRNA UAC AAA UUU AA Met Phe Lys STP

41

Codon-anticodon

The mRNA sequence complementary to the tRNA anticodon is called a codon

The sequence of aminoacids along a protein is specified by the anticodon-codon alignment

Alignment is anti-parallelIf anticodon is 3’CCU5’, complementary mRNA codon is 5’GGA3’

tRNA translate the sequence of nucleotides present in the mRNA into a sequence of amino acids in the protein.

42

Reading the genetic code

A T G T T T A A A T A G C C C

C A T A A A T T T C T A G G G

5’ 3’

5’3’

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

5’ 3’

DNA

RNA

43

No Gaps

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

5’ 3’

U A C

Met

A A A

Phe

U U U

Lys

S T P

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

5’ 3’

44

No overlaps

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

5’ 3’

U A C

Met

U U U

Lys

G G G

Pro

S T P

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

5’ 3’

45

Protein synthesis is a stepwise process

5’ 3’

aa1

aa2

aa2

5’ 3’

aa1

aa2

5’ 3’

aa1

aa2

5’ 3’

aa1

aa3

46

Enzymes are required for protein synthesis

Mixing mRNA with charged tRNA’s does not lead to protein synthesis

The enzyme necessary for catalysis of protein synthesis is the RIBOSOME

Ribosomes are complex enzymes made of more than 50 proteins and 3 RNA molecules

The RNA molecules in ribosomes are called ribosomal RNA (rRNA)

The Ribosome has 5 functional sites

mRNA binding site

P A

Peptidyl transferase

2 tRNA binding sites

47

STEPS

5’-----UUCUGG-----3’

AAG

MetAla

Leu

Phe

ACC

Trp

5’-----UUCUGG-----3’ACC

Trp

AAG

MetAla

Leu

Phe

5’-----UUCUGG-----3’

AAGACC

MetAla

Leu

Phe

TrpAAG

5’-----UUCUGGUUU--3’ACC

MetAla

Leu

Phe

Trp

AAA

Phe

48

Translation termination

The growing polypeptide chain is released when a stop codon is reached

There are three stop codons: UAA UAG UGA

These codons are not recognized by a tRNAThey are recognized by a protein- Release factor.

This causes the ribosome to release the mRNA and the newly synthesized polypeptide

5’-----UGGUAA-----3’ (mRNA)ACC

Trp

MetAla

Leu

Phe

The release factorbinds to the STOP codon

49

Translation Initiation

What about the first aminoacid?Does the ribosome start synthesis at the start of the mRNA?

Translation of an mRNA by the ribosome always initiates at the INITIATION Codon- AUG

AUG is normally recognized by a tRNA charged with the amino acid Methionine

When an AUG occurs near the 5’ end of the mRNA(at a special initiation position),it is recognized by a special tRNA charged with

N-formylmethionine = fMet

50

Special Initiation position

What is the special initiation positionMost mRNAs will have more than one AUG codons.How is the initiation codon specified?

Upstream (5’) of the start codon AUG is a sequence in the mRNA that is Complementary to a sequence in one of the ribosomal rRNAs

Pairing of the ribosomal RNA with the mRNA serves to align the ribosome with the mRNA

UCCUCCA- 5’-----AGGAGGU--AUGUCUAUGACC-----3’ (mRNA)

(rRNA)

51

Predicting Genes

If you sequence a large region of DNA, how do you determine if the region codes for a protein or not?

5’ 3’5’3’

1

2

3

4

5

6

0 100 200 300 400

Start/Stop method

5’ ATG GCC TAT GAG AAT TAA TGA CCC GGG --

5’ ATG GCC T ATG AGA ATT AAT GAC CCG GG--

Start codon = ATGStop codon = UAA UAG UGA

52

Predicting Genes

XXXXXXXXATGGATGGATGAATGAATGA

ATGGATGGATGAATGAATGAMetAspGlyStpATGGATGGATGAATGAATGA MetAspGluStpATGGATGGATGAATGAATGA MetAsnGluStp

The first amino acid in any and all proteins is always Met (ATG)

The end of a protein is specified by Stop codonsTAA TAG TGA

TCATTCATTCATCCATCCAT

Is there a ribosome binding site upstream of the ATG

Is there a promoter upstream of the ribosome binding site

53

Genes also require promoters and ribosome binding sites

---TTGACAT------TATAAT-------AT--AGGAGGT--ATG CCC CTT TTG TGA---AACTGTA------ATATTA-------TA--TCCTCCA--TAC GGG GAA AAC ATT

(-10)(-35)

PROMOTER

5’3’

3’ 5’

antisense

sense

RIBOSOMEBINDING

SITE

T--AGGAGGT--AUG CCC CUU UUG UGA

5’ 3’

Met Pro leu leu stp

Prokaryotic Genes

Eukaryotes are more complicated

54

Genes also require promoters and ribosome binding sites

---TTGACAT------TATAAT-------AT--AGGAGGT--ATG CCC CTT TTG TAA---AACTGTA------ATATTA-------TA--TCCTCCA--TAC GGG GAA AAC ATT

(-10)(-35)

PROMOTER

5’3’

3’ 5’

antisense

sense

RIBOSOMEBINDING

SITE

Structure of a gene

Structure of the mRNA

Structure of a protein

55

The Genetic Code

Properties of the Genetic code

1- The code is written in a linear form using the nucleotides that comprise the mRNA

2- The code is a triplet: THREE nucleotides specify ONE amino acid

3- The code is degenerate: more than one triplet specifies a given amino acid

4- The code is unambiguous: each triplet specifies only ONE amino acid

5- The code contains stop signs- There are three different stops

6- The code is comma less

7- The code is non-overlapping

8- The code is universal: The same “dictionary” is used by viruses, prokaryotes, invertebrates and vertebrates.

56

UUUUUCUUAUUG

CUUCUCCUACUG

AUUAUCAUAAUG

GUUGUCGUAGUG

UCUUCCUCAUCG

CCUCCCCCACCG

ACUACCACAACG

GCUGCCGCAGCG

UAUUACUAAUAG

CAUCACCAACAG

AAUAACAAAAAG

GAUGACGAAGAG

UGUUGCUGAUGG

CGUCGCCGACGG

AGUAGCAGAAGG

GGUGGCGGAGGG

Phe

Leu

Leu

Ile

Met

Val

Ser

Pro

Thr

Ala

Tyr

STOP

His

Gln

Asn

Lys

Asp

Glu

Cys

STOP

Trp

Arg

Ser

Arg

Gly

U

C

A

G

U C A G

UCAG

UCAG

UCAG

UCAG

Fir

st

lett

er

Second letter

Th

ird le

tter

The GENETIC CODE

57

The code

3 amino acids are specified by 6 different codons5 amino acids are specified by 4 different codons1 amino acid is specified by 3 different codons9 amino acids are specified by 2 different codons2 amino acids are specified by 1 different codons

The degeneracy arises because

More than one tRNA specifies a given amino acidA single tRNA can base-pair with more than one codon

tRNAs do not normally pair with STOP codons

----UCC------UCA------AGCAGG

Ser

AGU

Ser

UCG

Ser

----UCC------UCA------AGG

Ser

AGG

Ser