1. Relationship between Genotype and Phenotype
Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
amino acid
sequence
protein
function
phenotype
organism

2. Cut plasmid with an enzyme that recognizes restriction site in lacZ’ gene (codes for -galactosidase).
Cut donor DNA with same enzyme.
Transform bacterial cells with recombinant plasmid.
Cells with recombinant plasmid have defective -galactosidase and cannot convert X-gal into blue dye.

3. Recombinant phages are used to infect E
Recombinant phages are used to infect E. coli to produce many copies of the desired DNA sequence.
Central portion of l genome is not necessary for replication or packaging.
That section can be replaced by foreign DNA fragment that you wish to clone.
Up to 15 kb of DNA can be cloned into phage genome.
Recombinant DNA can be packaged into phage capsule or head.

4. Modes of delivering recombinant DNA into bacterial cells
(a) Plasmid DNA is introduced into host cell by transformation.
(b) Cosmids are introduced in phage heads by transduction. Once inside, they replicate as large plasmids.
(c) Phage vectors are introduced by infection.

5. Relationship between Genotype and Phenotype
Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
amino acid
sequence
protein
function
phenotype
organism

6. Four Ribonucleotides Found in RNA

8. 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.

9. 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.

10. Promoter Sequences in E. coli
Promoters signal transcription in prokaryotes.

11. Transcription Initiation in Prokaryotes
s subunit positions RNA polymerase for correct initiation.
Upon initiation of transcription, s subunit dissociates.

12. Elongation NTP + (NMP)n (NMP)n+1 + PPi
RNA polymerase adds ribonucleotides in 5’ to 3’ direction.
RNA polymerase catalyzes the following reaction:
DNA
Mg++
RNA polymerase
NTP + (NMP)n
(NMP)n PPi

13. 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.

14. RNA polymerase recognizes signals for chain termination.
(1) Intrinsic: Termination site on template DNA consists of GC-rich sequences followed by A’s. 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.

15. Colinearity of Gene and Protein
genotype
DNA
DNA sequence
transcription
RNA
translation
amino acid
sequence
protein
function
phenotype
organism

16. 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.

17. Relationship between Genotype and Phenotype
Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
amino acid
sequence
protein
function
phenotype
organism

18. * eukaryotic RNA is monocistronic prokaryotic RNA can be polycistronic
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

19. Eukaryotic RNA
Many proteins must assemble at promoter before transcription.
General transcription factors (GTF’s) 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.

20. Prokaryotic and Eukaryotic Transcription and Translation Compared

21. 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.

22. 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.

23. 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 adenine nucleotides is added.
Polyadenylation is another characteristic of transcription in eukaryotes.

24. 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.

25. 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.

26. 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.

27. Reactions in Exon Splicing

28. 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.