Regulation of Gene Expression Flashcards
DNA Transcription or RNA Synthesis
The single strand RNA product is complementary to the DNA template strand
RNA polymerase adds 40 nt / sec at 37 ̊C in E. coli
RNA polymerase is highly processive
RNA polymerase moves along the DNA forming a 12-20 base pairs transcription bubble
RNA pol will open DNA and then made it back
Transcription starts at promoters and stops at transcriptional terminators
Transcription generates 3 types of RNA:
-mRNAs(proteins)
-tRNAs(carry a.a. to the ribosome)
-rRNAs(16S , 23S, 5S(scaffold))
RNA structure
mostly single-stranded(5’-3’)
Secondary structure: Stem &Loops
RNA coils back on itself and makes complementary base pairing
Secondary structure fold into tertiary structure
The sugars contain an OH group at the 2’ position (ribose)
The nitrogenous bases are A, C, G and U
t RNA how many nucleotide
76
stop codon
UAA UGA UAG
Procaryotic Transcriptional Terminator
Rho: protein factor
Rho independent
–Hairpin in RNA strand
–6 uridines after the hairpin structure (weak link)
–No need for rho factor
Rho dependent
–Need the rho factor
–No poly(U) tract
–Sometimes no hairpins
hair pins stuck in RNA pol, Rho open base pair
Christmas tree of rRNA
RNA start fold before translation end
Eukaryotic mRNA
*Monocistronic (1 gene)
*Cap structure added at the 5’ end
*Poly(A) tail added at the 3’ end
*contains numerous introns
*transcription (nucleus)
*translation (cytoplasm)
Prokaryotic mRNA
*Multicistronic (operon) 1 gene encode many gene
*No Cap structure and Poly(A) tail
*In general, no introns (some exceptions)
*Transcription and translation are coupled (no nucleus)
The 5’ Cap of Eukaryotic mRNA
7-methylguanosine
methyl group modify the 2’ -OH
polysome
RNA covered by ribosome
Translation or protein synthesis
*The mRNA nucleotide sequence is translated into an amino acid chain (protein)
*The ribosome is the site of translation (900 residues / min; 15 a.a. / sec)
*Ribosomes are 80 nt apart on mRNAs (polysomes or polyribosomes)
*tRNAs bring the a.a. to the ribosome
*tRNAs (anticodons) read the mRNA codons
*The ribosomes synthesize proteins from the N-terminus (NH2) to the C-terminus (COOH)
3 steps in translation
Initiation
Elongation
Termination
Transfer RNA (tRNA)
*Between 73 and 93 nt
*Form a “cloverleaf” secondary structure
*The anticodon triplet is complementary to the codon triplet on mRNAs
*The different a.a. are attached to their corresponding tRNA at the 3’ end (aminoacyl-tRNA synthetases）
The E. coli ribosome
S sediment factor
small：30S （16S rRNA= 1600nt+21polypeptide chain）
large：50S （5S+23S+34 polypeptide）
total：70S
Protein synthesis
16S RNA base pair with mRNA bring 30s subunit to the RNA
30 s subunit find the start codon brings in the first tRNA(have methionin) with 50s subunit
2 site in Ribosome
P site: carry the growing chain
A site: new tRNA
a.a. Chain transfer from P site to A site
P site tRNA empty
ribosome move forward
tRNA move from A to P
Stop codon:
tRNA without a.a. binds to A Site, facilitate release of the poly peptide and the mRNA from ribosome
Regulation of mRNA Synthesis
1.Negative Control (repressor protein):
–Induction:The gene is turned on when the enzyme is needed (substrate present / inducer)
–Repression:The gene is turned off when the end product accumulates(corepressor)
ex. lactose metabolism(lactose is the inducer)
tryptophan
2.Positive Control(activator protein): Turned on only in the presence of a controlling factor
ex. lac operon
3. Attenuation
Initiation and continuation of transcription are controlled
Attenuation of tryptophan
5 gene
trpR P O
trpL
attenuator
Leader peptide sequence (only include sequence 1)
trp codons: site stuck ribosome
Genetic exchange
*Recombination:
rearrangement of nucleic acids to produce a new nucleotide sequence
*Homologous
Recombination:recombination between two similar (homologous) sequences
more homologous, more efficient recombination
*Crossing-Over:
exchange of homologous pieces of DNA
*Horizontal Gene Transfer:
transfer of genes or DNA pieces from one organism to another
DNA recombination in bacteria
*Homologous recombination (X-over)
*Nonreciprocal recombination: only one way, no exchange
*Site-specific recombination (viruses)
insert a viral pieces of DNA into the chromosome
*Replicative recombination (mobile genetic elements)
Crossing-over
RecBCD:
helicase
5’-3’ exonuclease
create single strand
Rec A like protein+accessory proteins:
recognize single strand
open another double strand
base pairing
form 2 holiday junctions:
can move along strand
DNA pol III +ligase:
fill the gap
2 results
resolution
resolvase
Nonreciprocal Recombination
open host gene
endonucleases
连起来
Bacterial Plasmids
*Small circular double-stranded DNA molecules (some are linear) ～1000bp
*Number of copy/cell varies from 1 to hundreds （由original replication 决定）
*Exist independently from the host chromosome (origin of replication)
*They contain few genes (not essential to the host but could be useful)
*Some can be found integrated into the host chromosome (episome)(e.g., Fertility factor)
Bacterial Plasmids Type
*Fertility factors(conjugative plasmids) (F factor)
*Resistance factors(antibiotic resistance genes, 1 to 8)
*Col plasmids
(bacteriocins, colicins)
*Virulence plasmids(produce toxins, resist to host defense)
*Metabolic plasmids (carry enzymes）
3 ways for Bacterial Gene Transfer
*Conjugation(require sexpilus, cell-cellcontact, plasmid transfer)
*DNA transformation (linear DNA or plasmids taken from the cellenvironment)
*Transduction (bacteriophages)
4 result of transfer
- integration of donor DNA
- donor DNA self-replicates （plasmid）
3.Donor DNA cannot self replicate
- Host restriction （directly degrade）
The Fertility Factor
*Conjugative plasmid (100 kb)
*Contains genes for cell attachment (sex pili) and plasmid transfer (tra region， 40 different genes codes for sex pili, pore synthesis, replication of the DNA)
*F+cell (cell containing an F plasmid and covered with pili)(donor cell)
*F-cell (no F plasmid)(recipient cell)
*This plasmid is an episome
*Contains its own origin of replication (oriV)
*Contains an origin of transfer (oriT)
*F’: have additional info on plasmid, such as a piece of gene from chromosome
*rolling circle replication from Ori T
transformation of DNA fragment from environment
DNA bind to membrane
endonuclease cut into small pieces（5-15kb）
enter membrane
exonuclease degrade one chain
single strand
if high homologous，stable transformation
if low or no homologous， unsuccessful transformation
Transformation with a plasmid
whole plasmid uptake by host cell
can replicate/integration
or
degrade
Phages
might bring a piece of chromosome to other cells
概率很低
abortive transduction
unsuccessful
stable gene transfer
