Transcript MolBioPrimer_2005-06

Molecular Genetics
A Quick Primer
Yin Li
2005-06-06
Harvard iGEM 2005
The Central Dogma
DNA (Genes)
Transcription
mRNA
Translation
Proteins
Yin Li
Harvard iGEM 2005
DNA
DNA nucleotide:
Single-stranded DNA (ssDNA): 5'-P vs. 3'-OH
Double-stranded DNA (dsDNA) has antiparallel strands
in a double helix.
Watson-Crick pairing (H-bonds):
Purines to pyrimidines:
Adenine = Thymine
Guanine Cytosine
Yin Li
Harvard iGEM 2005
RNA
RNA nucleotide:
2'-OH (ribose sugar) instead of 2'-H (as in deoxyribose)
Uracil instead of thymine
Three kinds:
Messenger RNA: carry genetic information from DNA
Ribosomal RNA: components of ribosomes, where protein
synthesis occurs
Transfer RNA: mediates pairing of mRNA information to
amino acids
Yin Li
Harvard iGEM 2005
Proteins
Amino acid:
Amino Group
Carboxyl Group
Functional Group: Polar, non-polar
Polypeptide: polymer of amino acids
N-terminus vs. C-terminus
4 Levels of Structure:
Primary Structure: amino acid sequence
Secondary Structure: alpha helices & beta sheets
Tertiary Structure: overall shape (conformation) of single
polypeptide
Quaternary Structure: multimeric (>1) proteins
Conformation defines function.
Yin Li
Harvard iGEM 2005
Genetic Code
Codon: a triplet of DNA or RNA nucleotides, encoding
one amino acid
Redundancy of the code
More than one codon for each amino acid
Last letter of the codon is often interchangeable (wobble)
Special codons:
Start codon (also methionine): ATG (AUG)
Stop codons: TAA, TAG, TGA (UAA, UAG, UGA)
Yin Li
Harvard iGEM 2005
DNA Replication
Semiconservative replication
1. Initiation:
A short RNA primer is first synthesized by an RNA
polymerase (primase)
2. Elongation:
dNTPs added to the 3'-OH by DNA polymerase
5' to 3' direction of elongation
Leading strand continuously replicated
Lagging strand discontinuously replicated
3. Proofreading
Yin Li
Harvard iGEM 2005
Mutations
Silent/synonymous: same amino acid
Missense: new amino acid
Nonsense: becomes stop codon
Frame shift: insertion or deletion of bases s.t. the codon
frame, when read from ATG, shifts
Yin Li
Harvard iGEM 2005
Transcription
1. Promoter recognition
RNA polymerase and transcription factors (TFs) need to
bind to promoter region for transcription to occur
Consensus sequences, e.g. TATA box
2. Chain initiation & elongation
RNA synthesis begins at transcription start site, next to the
promoter
Again: 5' to 3' elongation
3. Termination
Chain-termination sequence (often forms hairpin loop)
Yin Li
Harvard iGEM 2005
mRNA
Transcription produces a primary transcript
Serves as mRNA in prokaryotes
Processed in eukaryotes:
1.
2.
3.
5' 7-methyl-guanosine cap
3' poly(A) tail
Intronic splicing (exons vs. introns)
5' and 3' untranslated regions (UTRs)
Open reading frame (ORF) start at AUG and end in
the first stop codon
mRNA stability varies
Yin Li
Harvard iGEM 2005
Translation:
1.
Initiation:
Prokaryotes: ribosome-binding site (RBS) on mRNA is recognized by
small 30S ribosome
Eukaryotes: initiation complex (including small 40S ribosome) latches
onto 5' cap
2.
3.
Scanning (eukaryotes only): initiation complex scans 5'-3' for
the start codon AUG
Beginning of synthesis:
Prokaryotes: large 50S ribosome is recruited and nearby AUG is
recognized
Eukaryotes: initiation complex dropped, large 60S ribosomal subunit
recruited
4.
5.
Yin Li
Elongation: tRNAs are sequentially recruited
Termination: stop codon is recognized, special tRNA carrying
release factor (RF) binds with ribosome
Harvard iGEM 2005
The Central Dogma Revisited:
Information Transfer & Sites of Regulation
DNA
ATG
5'
3'
Promoter
TSS
TAC
Stop
Template DNA
Stop
3'
CTS
5'
Transcription
RNA Processing
mRNA
5'
5' UTR
AUG
ORF
Stop
3' UTR
3'
mRNA Degradation
Translation
Protein
N
C
Post-Translational
Modifications
Yin Li
Harvard iGEM 2005
Transcriptional Regulation
Promoter strength depends on
Consensus sequences within promoter (e.g. TATA box)
Eukaryotes: enhancer and silencer regions
Negative regulation: basal promoter activity ON, needs to be
turned OFF.
Inducible: repressor is normally active; inducer required to remove
repression.
Repressible: (apo)repressor is normally inactive; co-repressor required to
activate repressor
Positive regulation: basal promoter activity OFF; needs to be
turned ON.
Autoregulation:
Positive: protein product induces further transcription
Negative: protein product inhibits further transcription
Yin Li
Harvard iGEM 2005
The E. coli lac Operon of Jacob &
The components of lactose metabolism are adjacent genes
Monod
governed by a single promoter
(lacP):
lacZ: beta-galactosidase (hydrolyzes lactose)
lacY: lactose permease (permits lactose entry)
lacA: thiogalactoside transacetylase
lacI
Inducible negative regulation:
lacP
lacO
lacZ
lacY
lacA
lacI: product is a repressor protein that normally binds to the operator
(lacO) region of operon to inhibit transcription
The lactose isomer allolactose and synthetic analogues (e.g. IPTG) binds
to repressor and permits transcription
Positive regulation:
Cyclic AMP concentrations indirectly regulated by glucose metabolism
(s.t. high [glucose] results in low [cAMP])
crp: cAMP receptor protein (CRP), which, when complexed with cAMP,
binds to lacP and permits transcription.
Yin Li
Harvard iGEM 2005