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
Complementarity and Asymmetry in RNA Synthesis
Only one strand of DNA is used as template for
RNA synthesis.
RNA bases are complementary to bases of template
DNA.
Template strand is antiparallel to RNA
transcript.
3
RNA Polymerase
RNA polymerase in E. coli consists of 4
different subunits (see model below).
s recognizes the promoter. Holoenzyme is needed
for correct initiation of transcription.
RNA polymerase adds ribonucleotides in 5 to 3
direction.
A single type of RNA polymerase transcribes RNA
in prokaryotes.
4
Promoter Sequences in E. coli
Promoters signal transcription in prokaryotes.
5
Transcription Initiation in Prokaryotes
s subunit positions RNA polymerase for correct
initiation.
Upon initiation of transcription, s subunit
dissociates.
6
Elongation
RNA polymerase adds ribonucleotides in 5 to 3
direction.
RNA polymerase catalyzes the following reaction
DNA Mg RNA polymerase
NTP (NMP)n
(NMP)n1 PPi
7
Termination
Termination of transcription occurs beyond the
coding sequence of a gene. This region is 3
untranslated region (3 UTR), which is recognized
by RNA polymerase.
8
Termination
RNA polymerase recognizes signals for chain
termination.
(1) Intrinsic Termination site on template DNA
consists of GC-rich sequences followed by As.
Intra-molecular hydrogen bonding causes formation
of hairpin loop.
(2) rho factor (hexameric protein) dependent
These termination signals do not produce hairpin
loops. rho binds to RNA at rut site. rho pulls
RNA away from RNA polymerase.
rut site
In E. coli, this structure signals release of RNA
polymerase, thus terminating transcription.
9
Colinearity of Gene and Protein
DNA
genotype
DNA sequence
transcription
RNA
translation
amino acid sequence
protein
function
organism
phenotype
10
Genetic Code
Genetic Code is nonoverlapping. A codon
(three bases or triplet) encodes an amino
acid. Genetic Code is read continuously from a
fixed starting point. There is a start codon
(AUG).
There are three stop (termination) codons. They
are often called nonsense codons. Genetic Code
is degenerate. Some amino acids are encoded by
more than one codon.
11
Molecular Basis for Relationship between Genotype
and Phenotype
DNA
genotype
DNA sequence
transcription
RNA
translation
amino acid sequence
protein
function
organism
phenotype
12
Eukaryotic RNA
Three RNA Polymerases
RNA Polymerase I II III
Synthesis of rRNA (except 5S rRNA) mRNA, some
snRNA tRNA, some snRNA, 5S rRNA
eukaryotic RNA is monocistronic prokaryotic
RNA can be polycistronic
13
Eukaryotic RNA
Many proteins must assemble at promoter before
transcription. General transcription factors
(GTFs) bind before RNA polymerase II, while
other proteins bind after RNA polymerase II binds.
Primary transcript (pre-mRNA) must be processed
into mature mRNA. 1. Cap at 5 end
(7-methylguanosine) 2. Addition of poly(A)
tail 3. Splicing of RNA transcript
Chromatin structure affects gene expression (gene
transcription) in eukaryotes.
14
Prokaryotic and Eukaryotic Transcription and
Translation Compared
15
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.
16
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.
17
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.
18
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.
19
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.
20
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.
21
Reactions in Exon Splicing
22
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.