Molecular Basis for

1
Molecular Basis for Relationship between Genotype
and Phenotype
DNA
genotype
DNA sequence
transcription
RNA
translation
amino acid sequence
protein
function
organism
phenotype
2
Transcription Initiation in Eukaryotes
TATA binding protein (TBP), part of TFIID
complex, must bind to promoter before other GTFs
and RNA polymerase II can form preinitiation
complex (PIC). Phosphorylation of carboxyl tail
domain (CTD), the protein tail of b subunit of
RNA polymerase II, allows separation of RNA
polymerase II from GTFs to start transcription.
3
Cotranscriptional Processing of RNA
State of phosphorylation of CTD determines the
type of proteins that can associate with the CTD
(thus defining cotranscriptional process). 5
end of pre-mRNA is capped with 7-methylguanosine.
This protects the transcript from degradation
capping is also necessary for translation of
mature mRNA.
4
Cotranscriptional Processing
3 end of the transcript typically contains
AAUAAA or AUUAAA. This sequence is recognized by
an enzyme that cleaves the newly synthesized
transcript 20 nucleotides downstream. At the 3
end, a poly(A) tail consisting of 150 - 200
adenine nucleotides is added. Polyadenylation is
another characteristic of transcription in
eukaryotes.
5
Complex Patterns of Eukaryotic RNA Splicing
Different mRNA can be produced different
a-tropomyosin can be produced. Alternative
splicing is a mechanism for gene regulation.
Gene product can be different in different cell
types and at different stages of development.
6
Intron Splicing Conserved Sequences
exons - coding sequences introns
- noncoding sequences
Small nuclear ribonucleoprotein particles
(snRNPs) recognize consensus splice junction
sequence of GU/AG. snRNPs are complexes of
protein and small nuclear RNA (snRNA). Several
snRNPs comprise a spliceosome. Spliceosome
directs the removal of introns and joining of
exons.
7
Spliceosome Assembly and Function
Spliceosome interacts with CTD and attaches to
pre-mRNA. snRNAs in spliceosomes direct
alignment of the splice sites.
One end of conserved sequence attaches to
conserved adenine in the intron. The lariat is
released and adjacent exons are joined.
8
Reactions in Exon Splicing
9
Self-Splicing Reaction
RNA molecules can act somewhat like enzymes
(ribozymes). In the protozoan Tetrahymena,
the primary transcript of an rRNA can excise a
413-nucleotide intron from itself.
These self-splicing introns are an example of
RNA that can catalyze a reaction.
10
Transfer of Information in Prokaryotes and
Eukaryotes
What are the similarities? What are the
differences?
11
Colinearity of Gene and Protein
DNA
genotype
DNA sequence
transcription
RNA
translation
amino acid sequence
protein
function
organism
phenotype
12
Colinearity of Gene and Protein
The linear sequence of nucleotides in a gene
determines the linear sequence of amino acids in
a protein. Mutant alleles of trpA gene differed
in the position of the mutation at the DNA level,
which corresponded to position of amino acid
substitution in the gene product.
Colinearity of mutations and altered amino acids
in a subunit of tryptophan synthetase from E.
coli C. Yanofsky, 1967. Scientific American
13
Tryptophan Synthetase Activity
indole-3-glycerol phosphate
? subunit
glyceraldehyde 3-phosphate
indole
serine
? subunit
tryptophan
14
Molecular Basis for Relationship between Genotype
and Phenotype
DNA
genotype
DNA sequence
transcription
RNA
translation
amino acid sequence
protein
function
organism
phenotype
15
tRNA
Anticodon of a tRNA molecule recognizes and pairs
with an mRNA codon. tRNA contains modified
bases pseudouridine, methylguanosine,
dimethylguanosine, methylinosine, dihydrouridine.
16
Genetic Code
17
Aminoacyl-tRNA Synthetase Attaches Amino Acid to
tRNA
Aminoacyl-tRNA synthetase catalyzes the formation
of charged tRNA. There is an aminoacyl-tRNA
synthetase for each amino acid. The carboxyl end
of an amino acid is attached to the 3 end of the
tRNA.
18
Wobble Position
Some tRNA molecules can recognize and pair with
more than one specific codon.
Base-pairing between the 3 base of a codon and
5 base of an anticodon is not always exact.