Baumeister Proteinbiosynthesis DNA

1. transcriptional control (+transcriptional accessability)

2. RNA processing control

3. RNA transport and localization control

4. translation control

5. mRNA degradation control

6. protein activity control

Most important is the Sigma Factor at the bacterial promotor, it binds to the DNA at the -10 / -35 bases of the DNA (Pribnow Box)

Bacterial transcription is more commonly controlled by repressors than activators —> it needs absence of repressor

the polymerase needs a start Codon and the sequence to read (bacterial genes are often polycistronic)

The Trp operon

—> at high levels of tryptophan the AA binds to the repressor and thereby activates it, when activated it binds to the operater sequence at the promotor (Operon) and blocks the binding of the RNA polymerase

The Lac operon

—> the other way around with lactose it binds to a Lac repressor and therebye removes it from the Lac opterator, the proteins for lactose metabolism should only be produced when needed, if there is glucose (more efficient metabolism) the operon is off the Lac genes are not needed that is achived by a cyclic AMP dependent CAP (catabolite activator protein) which only binds when cyclic AMP is present (in absence of glucose), in combination of absense of glc and presense of lactose the Lac operon is active and the Lac genes transcribed

eukaryotic RNA polymerase II requires a set of general transcription factors

• Most eukaryotic promoters contain a DNA sequence called the TATA box (ca. -25).

• the TBP (TATA binding protein) a subunit of TFII D binds to TATA Box

• that enables binding of TFII B

• at this prepared binding site the rest TF and the RNA Polymerase II can assemble

• the TFII H acts as helicase and pries apart the double Strand and phosphorylates the RNA polymerase and releases it

  
  

RNA Polymerase I —> rRNA genes

RNA P II —> all protein coding genes, miRNA genes, noncoding RNAs (splicosome)

RNA P III —> tRNA genes, 5S RNA genes, small RNAs

• BRE

  • TATA

    • INR

      • DPE

these Elements all have consensus sequences (specific base patterns) and all have a transcription factor that uses them to as a binding site

• Pribnow Box vs TATA Box

• Sigma factor vs mutiple TF

• operon with operator (mostly repressor) between -35/-10 binding site vs enhancer or silencer sites that can also occur in distance and still regulate transcription by folding (mediator proteins)

• in Eukaryotes the RNA polymerase tail is phosphorylated to bind

  • capping factors (5`beginning of mRNA at start of transkription, adding a methylated Guanosin [GTP] protecting the mRNA from Exonucleases, marking it for translocation out of nucleus and initiating translation)

  • splicing factors (splicing of introns or alternative spicing intron and some exons)

  • polyadenlylation factors (marking of 3`tail as end of mRNA and hairpin formation)

• alteration of Gene expression

• specific expression of target genes in specific tissues by using the specific promotor and genetic engineering

• create hybrid DNA binding proteins consisting of Pro- and Eukaryotic proteins so we can have a DNA-binding domain from one and a activation domain from another organism

The Arrangement of Chromosomes into Looped Domains Keeps Enhancers in Check

• different co-activators assemble to form a binding site that interacts with MEDIATOR

• same for repressors that turn gene translation off

• mixed activators that have RNA molecules as binding partners between protein co activators

• one activator recuits another activator or at some point the RNA polymerase itself

• an activator can bind and trigger release of RNA polymerase to start transcription or stop a pause

• one activator can trigger one and another two transcription units but when both bind simultaniously they can trigger massive increase of 100 transcription units

• one activator can act on different genes

1. activator protein synthesis itself acts as regulation (it needs to be produced to activate)

2. ligand binding (glucocorticoid (hormone) receptor)

3. covalent modification (phosphorylation)

4. addition of 2cd subunit (complex building)

5. unmasking (removal of inhibitor e.g. by phosphorylation)

6. stimulation of nuclear entry (when entering nucleus inhibitor stays in cytosol)

7. release from membrane

extra: transcriptional regulators can recruit chromatin remodeling complex to grant better access to DNA (more euchromatin), histone chaperones freeing DNA from histones, histone change or modification

the general TF with mediator and RNA polymerase bind histone modifing enzyme and chromatin remodeling complex

histone modifing enzyme —> adds acetyl groups to specific histones, which can then serve as binding sites for proteins that stimulate transcription initiation

chromatin-remodeling complexes —> render the DNA packaged in nucleosomes more accessible to other proteins in the cell, including those required for transcription initiation; notice, for example, the increased exposure of the TATA box.

the more accessable the TATA box and the DNA the better the transcription works…

• histone core removal

• nucleosome (DNA+histone) moving to grant access

• histone change and histone modification (acetylation = open)

Leucine zipper proteins:

• dimerized - 2 alpha helices bound together by interactions between mostly leucine (hydrophobic AS) the Y shaped endings interact with DNA major groove liek a clothespin (Wäscheklammer)

related: Helix-turn-Helix proteins

• dimerized but the 2 helices are each interrupted and have a loop in them and then continue --> directional change

Beta-sheet DNA recognition protein

• 2 stranded beta sheet laying in the major groove its AS interact with the DNA

ZINC-finger Protein

• containing zinc atoms as structural components

Sex hormones are TF

on the same side as the gene

the cis-regulatory elements can be cooperative or non-cooperative (2 regulatory elements need to find eachother first (non-cooperative) and then bind together, cooperative elements are dimerized initially)

A master regulator can control whole cascades of reactions

if the cell is exposed to stress (heat shock or UV light) as a first response the Gen of a regulator protein in transcribed, then this regulator protein binds to the promotors of several other gens, that trigger emergency responses for the cell survival or even apoptosis

(very few transcription factors can start important cascades for example change the cell type -> eye in the leg of drosophila [TF: Oct,Sox,Klf])

With the DNase footprint:

mix DNA strand and the TF and then add DNase to randomly cut the strands, whereever a TF is bound its not cleaved in a gel electrophorese you can then see the parts where much less strands were cut

Chip-Seq

a bound TF disrupts the read (seqencing) when compared to reference genome

Bacterial Repressor: A typical bacterial repressor, such as the lac repressor in Escherichia coli, usually functions by binding to a specific DNA sequence known as the operator. The operator is positioned near/in the promoter region of the target gene. The binding of the repressor to the operator physically obstructs the RNA polymerase from binding to the promoter, preventing transcription initiation. The repression is often released when a small molecule, known as an inducer, binds to the repressor, causing a conformational change and releasing the repressor from the operator, allowing transcription to proceed.

Eukaryotic Repressors: Eukaryotic repressors operate in the more complex environment of the eukaryotic cell nucleus. Their mechanisms of action are diverse and can involve various interactions with DNA, other proteins, or chromatin structure. Here are three ways eukaryotic repressors can work:

1. Direct DNA Binding and Steric Hindrance: Eukaryotic repressors may bind directly to specific DNA sequences in the promoter regions of target genes. This binding can lead to steric hindrance, preventing the assembly of the transcriptional machinery or blocking the binding of activators. The repressor-DNA interaction can also recruit co-repressor complexes that modify chromatin structure, making the DNA less accessible for transcription.

2. Recruitment of Co-Repressor Complexes: Eukaryotic repressors often recruit co-repressor complexes, which contain enzymatic activities responsible for modifying chromatin structure and suppressing transcription. For example, histone deacetylases (HDACs) within co-repressor complexes can remove acetyl groups from histone tails, leading to a more condensed chromatin structure that restricts access to the transcriptional machinery.

3. Interference with Activator Proteins: Eukaryotic repressors can interfere with the function of activator proteins. Activators typically bind to enhancer regions of DNA and stimulate transcription. Repressors may compete with activators for binding to these enhancer elements or physically interact with activators, preventing them from initiating transcription. This competitive binding or physical interaction can modulate the balance between activation and repression.

To achieve neuron-specific expression of the APP::GFP fusion protein in mice, you can use a neuron-specific promoter or regulatory element to drive the expression of the transgene. The goal is to restrict the expression of the APP::GFP fusion protein to neurons, preventing its expression in other tissues or cell types.

1. Select a Neuron-Specific Promoter: Identify a neuron-specific promoter or enhancer that is active in the desired neuronal cell types. Neuron-specific promoters are sequences of DNA that drive gene expression specifically in neurons. Examples of neuron-specific promoters include the neuronal promoter elements from the Synapsin I, CamKII, or Thy1 genes. (Promotor aex-3 for neuron expression example in lecture)

2. Clone Neuron-Specific Promoter with APP::GFP: Clone the neuron-specific promoter upstream of the APP::GFP coding sequence. This construct, which includes the neuron-specific promoter, will drive the neuron-specific expression of the APP::GFP fusion protein.

a. Gene Polymorphism:

• A gene polymorphism is a naturally occurring genetic variation within a population. It refers to the presence of multiple alleles or sequence variants at a specific locus in the DNA sequence of a gene. Gene polymorphisms can include single nucleotide polymorphisms (SNPs), insertions, deletions, or other types of genetic variations.

• A gene polymorphism does not always interfere with the function of the affected protein because not all variations in the DNA sequence lead to changes in the encoded protein. Silent mutations, synonymous SNPs, or variations in non-coding regions may have no impact on protein function. Additionally, some polymorphisms may result in conservative amino acid substitutions that do not alter the overall function of the protein.

b. Reverse Genetics Approaches in Model Organisms (Drosophila or C. elegans):

1. RNA Interference (RNAi):

   • Method: Introduce double-stranded RNA (dsRNA) specific to the target gene (let-60/Ras) to trigger RNAi-mediated gene silencing.

   • Advantage: Quick and relatively simple. Allows for the knockdown of gene expression.

   • Disadvantage: Potential for off-target effects, and knockdown may not be complete.

2. Gene Knockout by CRISPR/Cas9:

   • Method: Use CRISPR/Cas9 technology to induce targeted double-strand breaks and create loss-of-function mutations.

   • Advantage: Precision in generating specific mutations. Enables the complete loss of gene function.

   • Disadvantage: Possibility of off-target effects, and the process may require optimization.

3. Transgenic Overexpression:

   • Method: Introduce an additional copy of the wild-type or mutant gene into the organism for gain-of-function studies.

   • Advantage: Allows the investigation of the effects of increased gene dosage.

   • Disadvantage: May lead to artificial overexpression effects and may not accurately reflect endogenous regulation.

4. Allelic Series or Mutant Allele Studies:

   • Method: Generate and study different alleles with varying degrees of functionality (e.g., hypomorphic alleles, missense mutations).

   • Advantage: Allows the assessment of the gene's dose-dependent effects on the phenotype.

   • Disadvantage: Generating a range of alleles may be labor-intensive, and the specific effects can be allele-dependent.

c. Additional Information for Cross-Species Analysis:

• Conservation of Amino Acid Residues: Identify conserved amino acid residues affected by the polymorphism in the human Ras gene and check if these residues are conserved in the homologous genes (let-60/Ras) of Drosophila and C. elegans.

• Functional Domain Conservation: Assess whether the polymorphism lies within functional domains that are conserved across species.

• Phenotypic Analysis in Model Organisms: Perform functional studies in the model organisms to examine whether the introduced mutation recapitulates the cancer-associated phenotype observed in humans.

• Complementation Assays: Conduct complementation assays by expressing the wild-type human Ras gene in model organisms carrying the mutation and vice versa to determine if the cross-species expression rescues or exacerbates the phenotype.

intestine: gly-19

muscle: unc-54

neuronal: aex-3 or rab-3

ubiquitous: cco-1 (if deleted = gene knockout)