Download CHAPTER 4, PART 2

CHAPTER 4, PART 2
FLOW OF GENETIC INFORMATION
CHAPTER 4, PART 2: FLOW OF GENETIC INFORMATION
LECTURE TOPICS
1. RNA properties in general
2. Transcription: RNA Polymerase
3. mRNA properties
4. Genetic Code
5. Eucaryotic genes: structural organization
RNA is usually a single-stranded polynucleotide
Single-Stranded DNA and RNA can form helical hairpins
Complementary when reversed (fold back and base pair)
3`
5`
3`
5`
5`
3`
5`
3`
Stem-loop (hairpin) structures
[Fig.5.19]
Typical highly base-paired RNA structure
* 3 bases form H-bonds
ends
*
[p.126 and Fig.5.20]
*
*
*
* *
(----) Extra H-bonds
(----) Watson-Crick H-bonds
in (G-C) base pair
Types of cellular RNA molecules
• All RNA in E. coli (a bacterium) is made by one RNA polymerase
• mRNA encodes protein amino acid sequences
The “Central Dogma” of molecular biology
10 -4,-5
10-3, -4
10-8
Transcription
translation
DNA
RNA
Replication
Reverse
transcription
DNA virus
Retrovirus
PROTEIN
RNA Virus
Prions
10-4
FEATURES OF PROCESSES
¾ Accuracy - RELATIVE
= error rates
¾ Signals - STARTS AND STOPS
¾ Stages - INITIATION, ELONGATION, TERMINATION
2) TRANSCRIPTION: DNA Æ RNA
RNA polymerase makes RNA that is
complementary to a DNA template strand
Coding strand DNA sequence is same as mRNA (or any RNA transcript),
but has T’s instead of U’s
TRANSCRIPTION: DNA Æ RNA
RNA polymerase: DNA-dependent enzymes which require:
A. A template DNA (preferably double-stranded)
B. Activated precursors (all 4 rNTPs – ATP, GTP, CTP, UTP)
C. Mg++
D.
Reaction is:
(RNA)n + rNTP
(RNA)n+1 + PPi
2Pi
E. Same reaction mechanism as DNA polymerase
F. RNA polymerase does not require a primer
RNA polymerase reaction mechanism:
(same as DNA polymerase) 3`-OH attacks α-P
3`
5`
Nucleophilic attack
3`
5`
New 3`Æ5` phosphodiester bond
• Sequences shown are DNA Coding Strand
• Sequences shown are same as RNA but T’s instead of U’s
TRANSCRIPTION START SIGNALS: Promoters
TRANSCRIPTION TERMINATOR: Stop Signal
“Signal” is in RNA transcript base sequence and structure
G-C rich “Stem” (D.S.)
5`
3`
Pyrimidines – usually U
3) mRNA (messenger RNA)
Jacob and Monod predicted properties of mRNA:
1. A polynucleotide (RNA)
2. Base composition complementary to a DNA template
3. Size variation reflects variety of protein sizes (3 bases/a.a.)
4. Transient association with ribosomes
5. Rapid turnover (~2 minute half life in E. coli)
T2 Bacteriophage used to learn mRNA properties
E. Coli
cell
After infection,
Cell makes:
1. DNA
2. RNA [FAST, after 1]
3. viral proteins
4. new virus
mRNA: Experiment to verify predictions
Known: T2 bacteriophage infection is followed by
synthesis of RNA, proteins, and new virus particles.
Experiment:
•
•
Infect E. coli with T2 phage (add 32P-phosphate at same time)
Study properties of polynucleotides that incorporate 32P-phosphate.
Proof that new RNA is complementary to T2 DNA
D.S. HYBRID
3H
3H 3H
1) HEAT
3H
32P
3H
3) COOL
+
2) Add
T2 DNA
32P
Result shows that T2 DNA has
genes that code for the new RNA
New RNA
mRNA
? What if you use E. coli DNA instead of T2 DNA?
T2 DNA
T2 Bacteriophage result:
mRNA: Base composition is complementary to DNA template
E. Coli
cell
After infection,
Cell makes:
1. DNA
2. RNA [FAST, after 1]
3. viral proteins
4. new virus
CHAPTER 4, PART 2: FLOW OF GENETIC INFORMATION
LECTURE TOPICS
1. RNA properties in general
2. Transcription: RNA Polymerase
3. mRNA properties
4. Genetic Code
5. Eucaryotic genes: structural organization
The “Central Dogma” of molecular biology
10 -4,-5
10-3, -4
10-8
Transcription
translation
DNA
RNA
Replication
Reverse
transcription
DNA virus
Retrovirus
PROTEIN
RNA Virus
Prions
10-4
FEATURES OF PROCESSES
¾ Accuracy - RELATIVE
= error rates
¾ Signals - STARTS AND STOPS
¾ Stages - INITIATION, ELONGATION, TERMINATION
Translation (RNA Æ Protein)
tRNA - adaptors that deliver a.a`s to the translation system
“transfer”
RNA
Base to a.a. code
* Codon (mRNA)
3` 2`
* Anticodon (tRNA)
Amino
Acid
Aminoacyl-tRNA
Base pairs with
mRNA codons
THE GENETIC CODE (1964)
1) 3 letters
2) Sequential
3) Non-overlapping
4) No “punctuation”
“Colinear”
Q? What if code read /BCD/EFG/….etc
THE GENETIC CODE
The Genetic Code: How many code words (codons)?
[43 = 64 codons of 3 bases each (all are used)]
[“The Universal Code”]
Arg = CG(N)
(Start) AUG
TRANSLATION: Start signals on mRNA
Mitochondrial Genetic Code has differences
[“Universal”]
*
*
GENETIC CODE: Summary
A. A codon (3 bases) specifies an amino acid
B. Sequential and non-overlapping
C. Degenerate (more than one codon/amino acid)
D. Some codons are start and stop signals
E. The code is nearly universal (see differences in
human mitochondrial code)
F. Sequences of bases in genes and amino acids in
their encoded proteins are colinear
G. Experiments with synthetic mRNAs established
codon assignments
CHAPTER 4, PART 2: FLOW OF GENETIC INFORMATION
LECTURE TOPICS
1. RNA properties in general
2. Transcription: RNA Polymerase
3. mRNA properties
4. Genetic Code
5. Eucaryotic genes: structural organization
Prokaryotic mRNA base paired to its gene
DNA-RNA hybrid,
base-paired
A continuous coding sequence on DNA
Eukaryotic mRNA base paired to its gene
β- globin mRNA-DNA hybrid
D.S. DNA
DNA-RNA
hybrid loops
Eucaryotic mRNA base-paired to its gene
(D.S. DNA)
(RNA-DNA
D.S. hybrid)
(D.S. DNA)
DNA coding sequence must be discontinuous!
Eukaryotic gene transcription and mRNA maturation
GENE
X1 X2
X3
X = exon
Pre-mRNA
X1 X2
X3
Removal of introns
+
mRNA
X1 X2
X3
All 3 exons joined
RNA intron splicing signals:
• Conserved in all eukaryotes.
• Occur at or near intron-exon junctions
A
5`
3`
Modified ends of Eukaryotic mRNAs
[Post-transcriptional]
N7-MeG
Protein
A mature mRNA
Why introns? One possibility is to facilitate
evolution of new proteins by exon shuffling
New genes code for proteins with a mix of exons from
pre-existing genes to get proteins with new functions.
Alternative Splicing can result in mRNA’s for different
proteins from same primary transcript
(Ex: membrane-bound vs soluble antibody)
1
2
3
Exon
Intron
1
2
3
4
4
5
A
Alternative
Splicing
5
Spliced
mRNAs
6
6 Exons
6
Pre- mRNA for
antibody gene
B
2
3
4
5 Exons
5
6
PROKARYOTE DNA
EUKARYOTE DNA
Exon
Intron Exon
Pre m-RNA
Splice out intron
m-RNA
m-RNA
Protein
Protein
Eucaryotic Genes:
Summary of Structural organization
1. Allmost all have coding sequences (exons) interrupted by noncoding sequences (introns)
2. After transcription, introns are removed and exons are joined
accurately by splicing at evolutionarily conserved sequences.
3. Exon polarity (5`Æ3`) is retained after splicing
4. Protein domains coded by exons can be rearranged to give
proteins with new functions
5. Alternate splicing of an mRNA can give different proteins. (Ex:
membrane-bound vs soluble antibody)
6. Almost all bacterial genes lack introns
(had but lost during evolution to adapt to very rapid growth)
CHAPTER 4, Part 2
Study Exercise
1. Draw representative prokaryotic and eukaryotic
genes
2. Indicate locations of signals for transcription and
translation, relative to each other
3. For a eukaryotic gene, indicate intron processing
signals and their location, relative to other signals