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DNA base pairs. Base pairing Anti parallel strands. Base pairing. DNA sequence (5 ’ to 3 ’ ) Gene sequence Intergenic sequence. Eukaryotic Information Transfer: Transcription & Translation. ****Beadle and Tatum: Gene = polypeptide****. DNA. Protein. - PowerPoint PPT Presentation
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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
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.
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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’
antisense
sense
What do we mean by conserved sequence?
Regions of the DNA (gene or non-gene) or protein that share similar nucleotide sequence
8
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)
9
Homology is never perfect
The sequence homology between genes is not usually perfectOnce all the genes are aligned, the most common residue ateach position is used to construct a consensus sequence
(15-17 bp)
Consensus sequences of promoters
----TTGACAT---------------TATAAA----------AT----ATG CCC
(-10)
(-35)
(+1)
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
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 Concensus
10
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.
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.
(15-17 bp)
Consensus sequences of promoters
----TTGACAT---------------TATAAA----------AT----ATG CCC
(-10)
(-35)
(+1)
11
RNA polymerase
RNA polymerase searches for the promoter
RNA promoter binds the promoterand unwinds the DNA
RNA polymerase synthesizes RNA
12
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
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RNA is synthesized in 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 the opposite direction (3’ to 5’)
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Promoters can be found in different relative orientations
15
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
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Transcription termination
5’ 3’
5’3’5’ 5’3’ 3’
Termination of transcription also requires specific DNA sequencesA protein, that associates with the RNA polymerase, recognizes this sequence This causes the polymerase to first pause and thendissociate from the DNA sense 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.
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Eukaryotic RNA
Prokaryotes have a single RNA polymeraseThis enzyme synthesizes mRNA, tRNA and rRNA
Eukaryotes have three RNA polymerasesRNA PolymeraseI----rRNARNA polymeraseII---mRNARNA polymeraseIII--tRNA
RNA is synthesized in the nucleusThis is the Primary transcriptIt 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
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Transcription Pre-Initiation Complex Formation
19
TATA
+/-
Upstream elements
These elements function to activate genes
20
InrTATAGene
Upstream element function
21
Properties of Enhancers (and Silencers)
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
Cooperative assembly of different factors
Integrity of the element depends upon all/most of the factors
Spacing of factor binding sites is important
22
Most enhancers function within open chromatin domains
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
- May aid in recruitment of enzymes that modify chromatin at the promoter
Mechanism of enhancer function
TATA
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Sequence specific proteins bind sites and open the chromatin domain
Architectural proteins help bend DNA via protein-protein interactions
Stable enhanceosome formed with a distinct protein surface
Enhanceosome surface interacts with complementary surface displayed by promoter bound complexes
Interactions induce recruitment of PIC factors and stabilize binding of the factorsAllowing multiple rounds of transcription
Model of enhancer function
24
Processing
DNA
Primary transcript
AAAA
Splicing
m1Gppp
m1Gppp
25
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
26
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
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SplicingShort 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
28
29
Mechanism of silencer function
Silencers generate inaccessible chromatin domains
Silencers prevent binding of transcription factors
Silencers prevent transcription
They recruit repressors
They recruit enzymes that modify chromatin
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
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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
31
TranslationTranslation 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?
32
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
33
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”
34
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
35
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
36
Charged tRNA
tRNA are synthesized from genes as RNA
A specific amino acid is covalently attached to the 3’ end of the tRNA by a specific AA-tRNA synthase (the true translators)
20 synthase enzymes for the 20 amino acids
This tRNA+AA is called a charged tRNAPro
37
tRNA genes, tRNA and charged tRNA
tRNA gene1 tRNA1UAC anticodon
Gene tRNAAA-tRNAsynthase Charged tRNA
Met-tRNAsynthase Met-tRNA
UAC
tRNA gene2 tRNA2AAA anticodon
Phe-tRNAsynthase Phe-tRNA
AAA
tRNA gene3 tRNA3UUU anticodon
Lys-tRNAsynthase Lys-tRNA
UUU
mRNA AUG UUU AAA UAA||| ||| |||
tRNA UAC AAA UUU AA Met Phe Lys STP
38
Codon-anticodonThe 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.
39
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’
DNA
A U G U U U A A A U A G C C C
5’ 3’
RNA
40
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’
41
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’
42
Protein synthesis is a stepwise process
5’ 3’
aa1
aa2
aa2
5’ 3’
aa1
aa2
5’ 3’
aa1
aa2
5’ 3’
aa1
aa3
43
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 several functional sites
mRNA binding site
P A
Peptidyl transferase
3 tRNA binding sitesE
44
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
45
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
46
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)
47
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
48
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)
49
Homology is never perfect
The sequence homology between genes is not usually perfectOnce all the genes are aligned, the most common residue ateach position is used to construct a consensus sequence
(15-17 bp)
Consensus sequences of promoters
----TTGACAT---------------TATAAA----------AT----ATG CCC
(-10)
(-35)
(+1)
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
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 Concensus
50
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
51
Predicting Genes
XXXXXXXXATGGATGGATGAATGAATGA
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
52
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
53
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.
54
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
55
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