central dogma
DNA is transcribed into RNA which is translated into proteins
gene
sequence of DNA or RNA that codes for a molecule that has a function
exon
actual protein coding region
intron
DNA sequence that is removed during RNA processing
only occurs in eukaryotes
5’-UTR and 3’-UTR
translation control
regions that are transcribed
exon
intron
5’-UTR
3-UTR
regions that are not transcribed
promoter terminator enhancer silencer etc...
promoter
special DNA sequences that direct RNA Pol to the initiation site
bound by RNA polymerase for starting transcription
terminator
transcription termination
enhancer and silencer
regulate gene expression
how many base pairs make up the human genome?
over 3 billion
coding DNA
sequences that can be transcribed into RNA and translated into proteins
makes up ~2% of total DNA
non-coding DNA
non-coding functional RNA cis- and trans-regulatory DNA sequences introns pseudogenes telomeres centromeres other repetitive sequences.... makes up ~98% of total DNA
non-coding functional RNA
many are critical elements in gene expression
types: ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA, snRNA, long non-coding RNA, etc.
cis- and trans-regulatory DNA sequences examples
promoters, enhancers, silencers, 5’-UTR, 3’-UTR
pseudogenes
inactive copies of protein-coding genes
are often generated by gene duplication
RNA polymerase
enzyme responsible for transcription
requires NTP and Mg2+
synthesis is 5’ to 3’
either DNA strand can be used as a template for transcription
does NOT require a primer
is NOT evolutionarily related to DNA polymerase
lacks 3’ to 5’ exonuclease activity
coding strand
DNA strand that has the same sequence as the RNA product (except T instead of U)
template strand
DNA strand that has a different sequence than the RNA product
how many subunits does bacterial RNA polymerase have?
5 (α, β, β’, ω, σ^70)
holoenzyme
made up of subunits: α (2), β, β’, ω, σ^70
is required for initiating RNA synthesis
core enzyme
made up of subunits: α (2), β, β’, ω
carries out the actual RNA polymerase activity
α subunit function
assembles core enzyme
interacts with regulatory factors
the C-terminal domain makes sequence-specific interactions at the UP element
contains determinants for interactions with regulatory factors
β subunit function
takes part in all stages of catalysis
β’ subunit function
binds to DNA — takes part in catalysis
ω subunit function
is required to restore denatured RNA polymerase into its native form
σ^70 subunit
takes part in promoter recognition (position RNA Pol for correct initiation)
decreases RNA Pol’s affinity for general DNA regions, which allows it to look for the promoter
makes sequence-specific contacts with the -10 and -35 regions
more than 1 is present in E. Coli
different ones recognize different promoters
stages of bacterial transcription cycle
initiation
elongation
termination
initiation
RNA Pol holoenzyme forms and locates a promoter – the polymerase unwinds the DNA at the place where transcription begins – transcription starts
elongation
once RNA polymerase has synthesized ~ 10 RNA nucleotides, the σ^70 releases and the polymerase shifts into elongation mode
rate is approx. 50 nucleotides/second
termination
when the polymerase encounters a termination signal, it leaves the DNA template and releases the RNA
upstream promoter element (UP)
40-60 nucleotides upstream of the transcription start site
is A/T rich
factors that affect the strength of a promoter
deviation from the consensus sequences
the distance between consensus sequences (17 bp is optimal)
transcription factors
transcription bubble
is formed when the DNA duplex is unwound over a short distance (normally ~17 bps)
backtracking
RNA Pol moves backwards when it becomes arrested
is a proofreading mechanism
strongly depends upon the stability of the RNA/DNA hybrid in the transcription bubble and the nature of the 3’-terminal residue (weaker hybrid = higher chance of arresting and backtracking)
intrinsic termination
the termination lies within the RNA transcript itself
intrinsic termination signal
the RNA product has a G/C rich palindromic sequence (which forms a hairpin), followed by several uracil residues
after the hairpin is formed: RNA Pol stalls, the RNA product is released, and the DNA double helix forms
Rho-dependent termination
termination depends upon the Rho protein
requires:
naked, unstructured DNA, no coupled translation, and slowed or paused transcription
Rho
hexameric helicase that specifically binds a stretch of 72 nucleotides on single stranded RNA (which is C-rich)
upon contact with the transcription bubble, it dissociates
negative regulation
involves repressors
ways negative regulation occurs
1) a repressor binds to the operator in the absence of a molecular signal – the external signal causes dissociation of the repressor to permit transcription
2) a repressor binds to the operator in the presence of a molecular signal – when the signal is removed, the repressor dissociates – transcription continues
positive regulation
involves activators
ways positive regulation occurs
1) an activator binds to the operator in the absence of a molecular signal – transcription proceeds; a signal is added – activator dissociates, transcription is inhibited
2) an activator binds to the operator in the presence of a molecular signal, it only dissociates when the signal is removed
operon
a cluster of related genes that share a promoter and regulatory sequences
the genes are transcribed together, so that one mRNA can encode several different proteins
is a common way that prokaryotic genes are organized
1st example: lac operon
lac operon
contains 3 genes for lactose metabolism: β-galactosidase (lacZ), lactose permease (lacY), and thiogalactoside transacetylase (lacA)
its expression is controlled by the lac repressor and glucose availability
β-galactosidase (lacZ)
cleaves lactose to from glucose and galactose
lactose permease (lacY)
transports lactose into the cell
thiogalactoside transacetylase (lacA)
transfers an acetyl group from acetyl-CoA to β-galactosidase (lacZ)
lac repressor
binds operators O1-O3 (primarily to operator O1), preventing RNA Pol from binding to the promoter
even with it present, transcription STILL OCCURS at a basal rate
dissociates from the operator when allolactose binds to it
has its own promoter
its transcription is independent of the transcription of the enzymes it regulates
is encoded by the gene lacI
requirements for the strongest induction of the lac operon
low [glucose]
high [cAMP]
lactose is present
trp operon
1st biosynthetic operon discovered
has 5 structural genes that encode enzymes for tryptophan synthesis
is regulated by repression and attenuation
trp repressor
binds to DNA in the presence of tryptophan
high [tryptophan] – tryptophan binds to the repressor – repressor associates with the operator – gene expression for tryptophan synthesis is slowed
attenuation
allows for a second level of regulation
responds to the concentration of charged tRNA^trp
is possible because in prokaryotes, transcription and translation occur simultaneously
riboswitch
regulatory segment of mRNA that binds to a small molecule, resulting in a change in the production of proteins encoded by the mRNA
most known ones occur in prokaryotes
can regulate gene expression at many different levels