1. DNA base pairs
Base pairing
Anti parallel strands

2. Base pairing
DNA sequence (5’ to 3’)
Gene sequence
Intergenic sequence

3. Eukaryotic Information Transfer: Transcription & Translation
****Beadle and Tatum: Gene = polypeptide****
DNA
Protein

4. RNA serves as the intermediary between DNA and proteins
Genes are in the nucleus
Proteins are made in cytoplasm
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

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

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 complex
It consists of four proteins in bacteria (E. coli)
A GENE is a defined region of DNA
It has
a start,
a body
a end.
Transcription involves THREE distinct processes
Transcription Initiation
Transcription Elongation
3) Transcription Termination

7. Initiation of Transcription
Initiation involves RNA polymerase recognizing
and binding to a specific sequence on the DNA
The recognition sequence is called a PROMOTER
PROMOTER 3’ 5’
sense
----TTGACAT TATAAT AT----ATG CCC GGG TTT TAA
----AACTGTA ATATTA TA----TAC GGG CCC AAA ATT
antisense 3’ 5’
(-35)
(15-17)
(-10)
The sequences are present in the promoters of most E. coli genes
These sequences are conserved
They are critical for proper functioning of the promoter
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 perfect
Once all the genes are aligned, the most common nucleotide at
Each position is used to construct a consensus sequence
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
(15-17 bp)
Consensus sequences of promoters
----TTGACAT TATAAA AT----ATG CCC
(-10)
(-35)
(+1)

9. Homology (molecular biology)
M A R T K Q T A R K S T G G K A P R K Q L A T mouse H3
M A R T K Q T A R K S T G G K A P R K Q L A T Dros H3
M A R T K Q T A R K S T G V K A P R K Q L A T Tetra H3
M 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
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)

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 replication
It is very likely that the human gene/protein will be
involved in replication

11. 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. The RNA polymerase holoenzyme contains an additional
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 promoter
and 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
--TTGACAT TATAAA AT--//-ATG CCC GGG TAA
--AACTGTA ATATTT TA--//-TAC GGG CCC ATT
template
Promoter sequence is asymmetrical and orients
the binding of the polymerase

15. RNA polymers are synthesized in the 5’ to 3’ direction
mRNA has the same sequence as the non-template strand
Once 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 termination5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’
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
TATA
Inr
TFIID
TFIIB
TFIIF
TFIIE
Polymerase
TFIIH
These factors bind promoters of ALL GENES

20. Eukaryotic RNA Prokaryotes have a single RNA polymerase
This 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 nucleotides long

21. Proximal promoter and Distal Enhancers
TATA +
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!

22. Upstream element function
Distal Enhancer
Proximal
Promoter
TATA
Inr
Gene
Transcription Activators bind the enhancer sequences and the promoter sequences. They cooperate together to activate transcription.
Activation domain
Helps recruit the general transcription factors
DNA binding domain
Each activator has a different domain that recognizes a different DNA sequence

23. Gene specificity Gal4 GAL1 PHO5 Galactose in media Gal4 GAL2 PHO8 PHO4
Phosphate in media
PHO4
GAL2
PHO8

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. Mechanism of enhancer function
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
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 SilencingAc 1
ORC
Rap1
Abf1 Ac 1 2 4
ORC
Rap1
Abf1 Ac 3 1 2 4
ORC
Rap1
Abf1 2 4 Ac 3 1
ORC
Rap1
Abf1 2 4 Ac 3 1
ORC
Rap1
Abf1

29. Processing
DNA
Primary transcript
m1Gppp
AAAA
m1Gppp
Splicing

30. Splicing Internal portions of the primary transcript are removed
This is called splicing
Regions of a gene that code for a protein are interrupted by
regions called intervening sequences (introns)
This was discovered by comparing the DNA sequence with the
mature cytoplasmic mRNA sequence 1 2 3 4 5 6 7
Gene
7700 nt
Ovalbumin
Capping, polyA
Splicing
mRNA
1872 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. Short sequences dictate where splicing occurs
Exon1
PuPuGUPuPu Py12-14AG
Exon2
Splice donor
Splice acceptor
Exon1
Exon2
Exon1
Exon2
Exon1
Exon2
Splicing requires a enzyme complex called a spliceosome
Consists 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

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
1958
Crick 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 acid
is 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 guess
One possibility more likely than any other -
they contain nucleotides”
“A separate enzyme would join each adaptor to each amino acid”

36. Adaptor hypothesis Proline GGG ||| AAACCCGGG
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

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 tRNA
Pro

40. tRNA genes, tRNA and charged tRNA
AA-tRNA
synthase
Gene
tRNA
Charged tRNA
tRNA1
UAC
anticodon
tRNA gene1
Met-tRNA
synthase
Met-tRNA
UAC
tRNA2
AAA
anticodon
tRNA gene2
Phe-tRNA
synthase
Phe-tRNA
AAA
tRNA3
UUU
anticodon
tRNA gene3
Lys-tRNA
synthase
Lys-tRNA
UUU
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-parallel
If 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 code5’ 3’
A T G
T T T
A A A
T A G
C C C
DNA
C A T
A A A
T T T
C T A
G G G 3’ 5’
A U G
U U U
A A A
U A G
C C C 5’ 3’
RNA

43. 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
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. A U G A A A C C C U A G 5’ 3’ U A C Met U U U Lys G G G Pro S T P
No overlaps
A U G
A A A
C C C
U A G 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 5’ 3’

45. Protein synthesis is a stepwise process5’ 5’ 3’ 3’
aa2
aa2
aa1
aa1 5’ 5’ 3’ 3’
aa2
aa2
aa3
aa1
aa1

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
Peptidyl transferase
2 tRNA binding sites P A
mRNA binding site

47. STEPS ACC Trp AAG Met Ala Leu Phe AAG Met Ala Leu Phe ACC Trp
5’-----UUCUGG-----3’
5’-----UUCUGG-----3’
Met
AAG
Ala
Leu
ACC
Met
Ala
Leu
Phe
Trp
Phe
Trp
AAG
ACC
AAA
Phe
5’-----UUCUGG-----3’
5’-----UUCUGGUUU--3’

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 tRNA
They are recognized by a protein- Release factor.
This causes the ribosome to release the mRNA and the
newly synthesized polypeptide
ACC
Trp
Met
Ala
Leu
Phe
The release factor
binds to the STOP codon
5’-----UGGUAA-----3’ (mRNA)

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
(rRNA)
UCCUCCA-
5’-----AGGAGGU--AUGUCUAUGACC-----3’ (mRNA)
What is the special initiation position
Most 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

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’ 3’ 5’
5’ ATG GCC TAT GAG AAT TAA TGA CCC GGG --
5’ ATG GCC T ATG AGA ATT AAT GAC CCG GG--
Start codon = ATG
Stop codon = UAA UAG UGA
Start/Stop method
100
200
300
400 1 2 3 4 5 6

52. XXXXXXXXATGGATGGATGAATGAATGA
Predicting Genes
The first amino acid in any and all proteins is always
Met (ATG)
The end of a protein is specified by Stop codons
TAA TAG TGA
XXXXXXXXATGGATGGATGAATGAATGA
TCATTCATTCATCCATCCAT
ATGGATGGATGAATGAATGA
MetAspGlyStp
MetAspGluStp
MetAsnGluStp
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
Prokaryotic Genes
PROMOTER 3’ 5’
antisense
---TTGACAT------TATAAT AT--AGGAGGT--ATG CCC CTT TTG TGA
---AACTGTA------ATATTA TA--TCCTCCA--TAC GGG GAA AAC ATT
sense 3’
(-35)
(-10)
RIBOSOME
BINDING
SITE 5’ 5’ 3’
T--AGGAGGT--AUG CCC CUU UUG UGA
Met Pro leu leu stp
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’
antisense
sense
RIBOSOME
BINDING
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
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. The GENETIC CODE Second letter U C A G UUU UUC UUA UUG CUU CUC CUA CUG
AUC
AUA
AUG
GUU
GUC
GUA
GUG
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
UAU
UAC
UAA
UAG
CAU
CAC
CAA
CAG
AAU
AAC
AAA
AAG
GAU
GAC
GAA
GAG
UGU
UGC
UGA
UGG
CGU
CGC
CGA
CGG
AGU
AGC
AGA
AGG
GGU
GGC
GGA
GGG U C A G
Phe
Tyr
Cys U
Ser
STOP
STOP
Leu
Trp
His C
Leu
Pro
Arg
Gln
Third letter
First letter
Asn
Ser A
Ile
Thr
Lys
Arg
Met
Asp G
Gly
Val
Ala
Glu

57. The code 3 amino acids are specified by 6 different codons
1 amino acid is specified by 3 different codons
9 amino acids are specified by 2 different codons
2 amino acids are specified by 1 different codons
The degeneracy arises because
More than one tRNA specifies a given amino acid
A single tRNA can base-pair with more than one codon
tRNAs do not normally pair with STOP codons
AGG
Ser
AGU
Ser
UCG
Ser
AGG
Ser
AGG
Ser
----UCC------UCA------AGC
----UCC------UCA------