MCB chapter 8

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ubiquitin ligases

(E1, E2, E3) Proteins targeted for degradation are ubiquitinated by regulatory enzymes called ubiquitin ligases Ubiquitin is 76 a protein that is attached to Lys side chains of a target protein § Ubiquitin can be thought as a way the cell tags certain proteins for recycling

Operons

A cluster on a chromosome and are transcribed from a single promoter as one long mRNA molecule Common in bacteria but rare in eukaryotes where genes are transcribed and regulated individually.

Transcriptional Repressor

In its active form it switches genes off, or represses them.

Transcription Regulators

Regulatory DNA sequences do not work by themselves but the sequences need to be recognized by these proteins. o It is the binding of a transcription regulator to a regulatory DNA sequence that acts as the switch to control transcription.

Differentiation

The ability to specialize into something

in absence of lactose

The lac repressor shuts off the operon in the absence of lactose. § In the presence of lactose, the repressor undergoes a conformational change and can no longer bind to the operator (allosteric inhibition).

upstream region from the promoter

This upstream region contains sites that are required for the RNA polymerize to recognize the promoter and sequences with recognition sites for proteins that associate with the active polymerase: sigma factor in bacteria or the general transcription factors in eukaryotes.

Operator

Within the operons promoter is a short DNA sequence called Operator, that is recognized by a transcription regulator

eukaryotic mRNA translation start mechanism

· mRNAs poses a 5' cap that helps guide the ribosome to the first AUG, the codon where translation will start. · Eukaryotic repressor proteins can inhibit translation by binding to specific nucleotide sequences in the 5' untranslated region of the mRNA, thereby preventing the ribosome from finding the first AUG.

Gene Expression

A complex process by which cells selectively direct the synthesis of the many thousands of proteins and RNAs encoded in their genome

Regulatory DNA sequences

Both bacterial or eukaryotic have these, that are used to switch the gene on or off. relatively short in bacteria and longer in eukaryotes (integrating information from a variety of signals into a command that dictates how often transcription gene is initiated)

master regulations

Cascade of regulators that work in combination, lead to formation of an organized group of many different types of cells.

Lac Operon

Encodes proteins required to import and digest the disaccaharide lactose.

Post-Transcriptional Regulation/Controls

One gene can encode for more than one type of protein § Splice together different exons

in absence of glucose

The bacterium makes cAMP which activates CAP to switch on genes that allow the cell to utilize alternative sources of carbon-including lactose.

Alternative Splicing

method of Post-Transcriptional Regulation Different combinations of exons are spliced together to produce different mRNAs à different proteins.

The Expression of Different Genes can be Coordinated by a Single Protein

As long as different genes contain regulatory DNA sequences that are recognized by the same transcription regulator, they can be switched on or off together, as a coordinated unit.

Transcriptional Activator

Switch genes on or activate them, work on proteins that are only marginally able to bind and position RNA polymerase on their own These poorly functioning promoters can be made fully functional by activator proteins that bind nearby and contact the RNA polymerase to help initiate transcription. § Often have to interact with second molecule to be able to bind DNA.

MicroRNAs (miRNAs)

Tiny RNA molecules that control gene expression by base-pairing with specific mRNAs and reducing both their stability and their translation into protein.

B Galactosides

an enzyme that breaks down lactose

Lac Operon in E. coli controlled by

both Lac Repressor and CAP activator.

The let-7 miRNA

negatively regulates the mRNA's of genes that are commonly overexpressed in cancer cells

Positive Feedback Loop in cell memory

A master transcriptase regulator activates transcription of its own gene, in addition to that of other cell-type-specific genes. · Each time a cell divides the regulator is distributed to both daughter cells, where it continues to stimulate the positive feedback loop.

Mediator

Additional proteins serve to link the distantly bound transcription factors to these proteins at the promoter, most important is mediator. Mediator forms a bridge between transcriptional Activators/Repressors and RNA Polymerase 2

Epigenetic Inheritance

All of these cell-memory mechanisms transmit patterns of gene expression from parent to daughter cell without altering the actual nucleotide sequence of DNA, considered to be forms of Epigenetic Inheritance.

How do Bacteria adapt so quickly to their environments

Bacteria regulate the expression of many of their genes according to the food sources that are available in the environment

EXAMPLE of Transcriptional Activator

Bacterial activator protein CAP has to bind cyclic AMP (cAMP) before it can bind to DNA. o Genes activated by CAP are switched on in response to an increase in intracellular cAMP concentration, which rises when glucose the bacterium carbon source, is no longer available. As a result CAP drives the production of enzymes that allow the bacterium to digest other sugars.

Why negatively regulate mRNAs with miRNAs instead of simply not transcribing them in the first place?

Don't want it to affect the cells that are healthy, microRNAs are a genome defense in a way

gene expression in prokaryotes

Gene expression will occur at both transcription and translation but more emphasize on transcription level because right after transcription occurs translation begins.

Chromatin Modifying Genes:

In eukaryotic cells, activator and repressor proteins exploit chromatin structure to help turn genes on and off. Nucleosomes remodeling makes promoters more accessible to transcription factors. ATP driven § Chromatin structure can be altered by chromatin-remodeling complexes and by enzymes that covalently modify the histone proteins. § Many Gene activators take advantage of these mechanisms and recruit chromatin modifying proteins to promoters.

Precurser miRNA transcript undergoes a special type of processing to yield mature miRNA.

It is packaged with specialized proteins to form RNA-induced silencing complex (RISC) which patrols the cytoplasm in search of mRNAs that are complementary to the bound miRNA molecule

gene expression in eukaryotes

Much more complex than prokaryotes. Many more steps between translation and transcription, so many more steps to regulate gene expression at.

RISC

Once a target mRNA forms base pairs with the miRNA it is either destroyed immediately by a nuclease present within the RISC or translation is blocked.

Cell Memory

Proliferating cell to maintain its identity. The patterns of gene expression responsible for that identity must be remembered and passed on to its daughter cells through all subsequent cell divisions.

Promoter

Region of gene that binds the enzyme RNA polymerase and correctly orientates the enzyme to begin its task of making an RNA copy of the gene. o The promoters of both bacterial and eukaryotic genes include a transcription initiation site, where RNA synthesis begins, plus a sequence of approximately 50 nucleotides pairs that extends upstream from the ignition site. Promoters are always adjacent to the coding sequence it controls § Promoters are always upstream of the coding sequence it controls § Promoter activity is directional, in inverted promoter will not function. Why always has arrow.

RNA Interference (RNAi)

The system is used to eliminate foreign RNA molecules, especially the double stranded RNAs produced by many viruses and transposable genetic elements. in some organisms the RNAi defense response can spread fro tissue to tissue allowing the entire organism to become resistent to the virus after only a few of its cells have been infected

Combinational Control

The way that groups of transcription regulators work together to determine the expression of a single gene.

tryptophan repressor

Transcription Regulator that can only bind to DNA if it has also bound several molecules of tryptophan

Process of Repression of Tryptophan Operon

When the regulator binds to the operator, it blocks access of RNA polymerase to the promoter, preventing transcription of the operon and production of the tryptophan producing enzymes.

Pax6

a TF that serves as a master regulator of eye development: The fly Pax6 gene is called eyeless Ey functions like any other transcription regulator, controlling regulatory regions. Some of the genes controlled by Ey encode additional transcription regulators that, in turn, control the expression of other genes. Misexpression of eyeless causes ectopic eyes § Misexpression Experiment: The eyeless coding sequence is expressed from a promoter/enhancer sequence from a gene expressed in a leg.

26S Proteasome

a protein complex found in all Eukaryotes and Archaea, and some bacteria. o The major function of the Proteasome is a Protease o Degrades: § Mis-folded proteins § Proteins that are regulated § Proteins are no longer needed § Cleaved protein fragments

histone deacetylases

an enzyme that remove the acetyl groups from histone tails, therefore reversing the positive affects of the acetylation has on transcription ignition.

Small interfering RNAs (siRNAs)

double stranded foreign RNAs are cut into short fragments by a protein called a dicer the resulting double stranded fragments are called siRNAs and are taken up by the RISC complex for degradation

bacterial mRNA translation start mechanism

mRNAs contain short ribosome binding sequence located a few nucleotide pairs upstream of the AUG codon where translation begins. · This binding sequence forms base pairs with the RNA in the small ribosomal subunit, correctly positioning the initiating AUG codon within the ribosome. By blocking or exposing the ribosome-binding sequence the bacterium can either inhibit or promote the translation of mRNA.

recruitment of histone acetyltransferaces

promotes the attachment of acetyl groups to selected lysins in the tail of histone proteins, this modification alters chromatin structure and allows access to underlying DNA.

Polycistronic mRNA

single transcript of DNA containing more than one coding sequence is translated into more than one polypeptide

Enhancers/Silencers

the DNA sites to which eukaryotic gene activators bind, their presence dramatically enhances the rate of transcription. § Can enhance transcription even when they are bound thousands of nucleotide pairs away from genes promoters. § Both transcription factors (activators and repressors) can bind to enhancers. § Also work when bound upstream or downstream from genes promoter.

Permease

transports lactose into the cell

mRNAs have three different fates once exported from nucleus

§ Either degraded by ribonucleus on 3' end, can not be chewed on by having a Poly A tail (Have not Poly A but other proteins that mark for degradation) § Actively translated (going to have initiation factors and Poly A protein) § Stored, just being saved for later. (Other proteins mark for storage.)

Methylation of DNA

· In Vertebrate cells, DNA methylation occurs at certain cytosine bases. · This covalent modification turns off genes by attracting proteins that bind methylated cytosins and block gene transcription. · DNA methylation patterns are passed on to progeny cells by the action of an enzyme that copies the methylation pattern on the parent DNA strand to the daughter DNA strand as it is synthesized.


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