Lecture 5: Prokaryotic Gene Regulation- Lac Operon Flashcards
Transcriptional regulation in bacteria is relatively simple because it
involves a single activator or repressor protein
Transcriptional regulation in eukaryotes is complex because (3)
- More complex transcriptional regulation: Multicellular eukaryotes use transcriptional regulation to create different cell types
- RNA processing of eukaryotic mRNA: extensive processing of eukaryotic mRNA
- Nuclear membrane: In eukaryotes, transcription and translation take place in different cellular compartments (transcription in nucleus, translation in cytoplasm)
Prokaryote have a —- of RNA polymerase
single type
RNA polymerase
Structural
composed of a 5-subunit core plus a subunit called sigma factor.
Sigma factor
Positions RNA polymerase corectly at the promotor and after transcription is initiated. sigma factor leaves. Some sigma factor recognize promotors of genes required for growth in high heat while others for nitrogen saturation
Factor independent termination
GC rich DNA followed bu an A rich stretch causes RNA polymerase to release from the DNA template. Forms hairpin loop followed by bunch of UUUU mrna. Very unstable and polymerase try to stable by stoping but hairpin makes it fall off.
Rho dependent termination
Rho factor recognize a C-rich sequence of RNA and climbs up to release RNA polymerase (stopped at terminator) from the DNA template.
Promotor
DNA sequence where RNA polymerase binds to initate transcription
Activator
What it is
Protein bringing the RNA polymerase closer to the promotor
Repressor (2 types)
Protein blocking RNA polymerase from binding to the promotor or blocking it from moving along the DNA
Allosteric effectors
Small molecules that bind to the allosteric site of regulatory proteins
Effector binds which allows the activator to
Bind at the activator binding site
Operon (4)
What it isx2+genes+product
- Linked genes under the control of a single promotor
- Functionally related genes (ex: all involved in lactose metabolism)
- Genes are transcribed onto a single mRNA molecule
- Either all or none of the gene products will be synthesized
Lac Operon (2)
WHat it is+ ecoli
- Operon required for the transport and metabolism of lactose
- Ecoli use glucose as their source of Carbon and energy but can use lactose when glucose is carce in the environment.
When lactose is present, Ecoli …
expresses the enzyme: B-galactosidase
Permease (Y)
Transports lactose into the cell
Beta-galactosidase (Z)
Modifies lactose into allolactose and cleaves lactose
I gene
encodes the repressor (Protein that can then bind to the operator)
Talk about the location of the I gene
it can be far away fro the lac operon or near, the location isnt important because only the protein that it encodes is important (free to float in cell)
Trans-acting
Affect gene expression of distant genes (on DNA other than where the trans-acting factor is encodoes)- in addition to nearby genes
Far away gene, nearby gene, gene on same DNA, on different DNA
Cis-acting
Affect gene expression of nearby genes (only genes on the sane piece of DNA as the cis-acting factor is found)
Affect nearby gene or gene on the same piece of DNA
Lac operon expression is regulated by both
negative regulation and positive regulation
Negative regulator
involves a repressor -> turning the lac operon ON/OFF
Positive regulation
involves an activator -> fine tuning expression levels from the Lac operon
Z and Y enzymes
get expressed at the same time and at the same level since they are on the same piece of mRNA
P-
RNA plymerase cannot bind to the promotor
Z-
Inactive Z
Oc
Cannot bind to operator
Constitutive mutation
operon is on wether the inducer is present or not
Z+ and Z-
Z+ is dominant over Z-
O mutation is
cis acting
I+ ad I-
I+ is dominant over I-
If glucose is present then expression from lac operon is
minimal
If there is no glucose then expression from lac operon is
enhanced
In the abscence of Lactose:
- Repressor binds to the operator blocking mRNA polymerase from processing forward (Ecoli doesnt need to make the Z, Y enzymes)
In the prescence of Lactose:
- Inducer binds to repressor causing a structural change that reduces the repressor’s affinity for the DNA (specifically the operator)
- RNA pol isnt blocked and free to go on and make mRNA
Allolactase
The inducer, a side product of lactose metabolism
RNA Pol II
Transcribe mRNA, SnRNA
Tata box (2)
what it is+where
- only in eukaryotic cells, stabilizes RNA polymerase II
- Approximately -25 position
Initiator element (Inr)
approximately +1 position
Enhancer (3)
What it is+ how it works+ found
- Transcriptional activator proteins bind to enhancer regions distant from the promotor to cause DNA looping bring mediator and RNA polymerase to the promotor resulting in high level of transcription. (Highly expressed genes)
- DNA sequences that can promote transcription
- Can be found very far away from the transcription start site (1000s of nucleotides)
Transcription factors
- Proteins that bind to DNA (recognize promotor sequences) and guide RNA pol II to the transcription start site
Transcription factor binding at enhancers allow:
- High level of transcription
- Large protein complexes interact with RNA pol II to turn transcription “ON” (many transcription factors are expressed in only specific tissues)
Mediator protein
Acts as a bridge between transcription factors and RNA pol II
17B-Estradiol (6)
What it is+ property+ steps
- a hormone
- Hydrophobic: Can diffuse across cellular membrane
- Binds to nuclear hormone receptor (soluble proteins on inside of cell). These receptors are located in the nucleus of target cells.
- When 17β-estradiol binds to estrogen receptors, it induces a conformational change in the receptor, leading to the formation of an estrogen-receptor complex.
- The estrogen-receptor complex acts as a transcription factor. It can bind to specific DNA sequences called estrogen response elements (EREs) located in the promoter regions of target genes.
- Hormone receptor complex binds to DNA elements to regulate gene expression
Co Activators
Proteins that facilitate transcription but do not bind to DNA directly.
5’ end of RNA transcript is modified during transcription: (2)
What it is + structure
- 5’ CAP (7-methylguanylate) protects the 5’ end of the mRNA from phosphatases and nucleases
- consists of a guanine nucleotide connected to mRNA via an unusual 5′ to 5′ triphosphate linkage. This guanosine is methylated on the 7 position
3’ end of RNA transcript is modified after transcription has ended (2):
how it ends+what happens
- Endonuclease recognize the cleavage signal, cleaving the mRNA transcript ending transcription
- Poly A polymerase adds up to 250 adenine nucleotides to the 3’ end (stability+translation efficiency)
Exon
- Sequence that encodes amino acids (proteins)
- Coding region
Intron
- Sequence that does not encode amino acids (protein)
- Noncoding region
Bacterias (prokaryotes) do not have
intron
Splicing
Introns are removed from pre-mRNA and exons are linked together to form the final mature mRNA
splicesome (5)
made up of+RNA+catalytic center+Need
- made up of 5 noncoding RNAs (SnRNA) complied to several proteins (snRNP) to form a ribonucleoprotein
- RNAs guide the complex to the splice site on the pre-mRNA
- RNA catalyze transeterification reactions
- snRNA U2 and U6 form the catalytic center of the spliceosome
- Need ATP to function (hydrolysis)
Genectic code
- groups of 3 nucleotides (bases) encode an amino acid (3codon)
- Has directionality, read 5’ end of mRNA to 3’ end
- Code is degenerate: most amino acids are encoded by more than 1 codon
Stop codon
Doesnt encode anything
What defines the reading frame?
The start codon (AUG)
tRNA (4)
What it does+ attached+ structure
- Transfer RNA, adaptor molecule between the codon and its specified amino acid
- Have a specific amino acid attached
- Has an anticodon loop that recognizes the codon in the mRNA sequence
- 3’ end of the tRNA is the acceptor stem that has an amino acid attached to the 3’ OH
Ribosome
- contains rRNA, coordinate interactions of tRNA. mRNA and facilitates peptide bond formation between amino acid
How are amino acids attached to tRNA?
- The amino acid must be activated before it is transferred to the correct tRNA molecule
- Activation and transfer are both accomplished by the enzyme Aminoacyl-tRNA synthetase
- Aminoacyl-tRNA synthetase links carboxyl group of amino acid to AMP using ATP to form aminoacyl-AMP
- Aminoacyl-tRNA synthetase transfer the aminoacyl-AMP to the correct tRNA molecule and AMP gets released
Aminoacyl-tRNA synthestase specificity
There is one specific aminoacyl-tRNA synthetase for each amino acid (20) and they must recognize the correct amino acid and the correct tRNA molecule/
Ribosome (4)
structure+catalytic+ translate
- Large subunit (50s) and small subunit (30s)
- rRNA makes up almost 2/3 of the subunit’s mass (noncoding RNA molecule)
- Key catalytic sites in the ribosomes are mostly RNA
- Ribosoes translate mRNA in the 5’-3’ direction
mRNA is bound by the —– subunit. tRNA interact with ——
- 30s
- Both the small and large subunit
Shine-Dalgarno sequence (2)
- in bacterial
- sequenc eon mRNA help to position the ribosome for translation initatiation
Kozak sequence (2)
- in eukaryotes
- sequenc eon mRNA help to position the ribosome for translation initatiation
Explain how protein synthesis in bacteria starts (3)
Starts with+ ribosome+ ecoli
- starts with the modified amino acid N-Formylmethionine (fMet)
- Inititator tRNA brings fMet to the ribosome to initate translation
- In Ecoli, it is not regular methionine that gets added as first amino acid
Formation of the 70s inititation complex: (3)
- rate limiting step in protein synthesis
- Involves protein initiation factors that hydrolyze GTP
- Establish the reading frame by binding to shine Dalgonra sequence
Translation elongation how it starts (2)
- Charged tRNAs are delivered to the A site of the ribosome by elongation factor Tu (EF-Tu) and requires GTP for binding
- EF-Tu does not interact with fMet-tRNA as fancy tRNA goes directly to the p-site and the rest of tRNA enter at A-site
Translation elongation: the process (3)
catalyzed by+ attack+ translocation
- Peptide bond formation is catalyzed by the peptidyl transferease center (part of the large subunit)
- Amino group of the aminoacyl-tRNA in the A site makes a nucleophillic attack on the esterlinkage between the initator tRNA and the formylmethionine in the P site
- Aided by EF-G which uses GTP hydrolysis to displace peptidyl-tRNA in the A site to the P site
New amino acid are added to
the carboxyl end of the growing peptide
Translation termination
- No tRNA anticodons recognize stop codons (UAA, UAG, UGA)
- Stop codons are bound by release factors that stimulate the cleave of the linkage between the tRNA and the polypeptide chain
