Dia-MCB181 Exam 3

Binding with a small molecule results in a conformational change in one part of the protein that initiates a chain of interactions and alters its structure. These changes may conceal amino acid side chains responsible for the original DNA-binding specificity and expose a new combination of amino acid side chains that a have a different DNA-binding specificity.

How can the binding of a molecule as small as a single amino acid change the ability of a large, multi-subunit protein molecule to bind a specific DNA sequence?

A mutation in this means that the repressor will always be able to bind with the lactose operon promoter. This will lead to a cell that is unable to produce b-galactosidase with or without lactose. Lactose operon will not be inducible.

What would be the consequence a mutation in the lacI repressor gene that produces repressor protein that is able to bind to the operator but is not able to able to bind allolactose?

-helps regulate the order in which successive carbs are utilized by the bacterium.

-in the absence of glucose, cAMP levels are high. CRP-cAmp binds to a site near the promoter of the lac operon and activates transcription

-with glucose, cAMP levels are low, CRP-cAMP is not formed and cannot bind so transcription is not induced to high levels

What is the role of the CRP-cAMP complex in positive regulation of the lactose operon in E.COLI?

Lactose Operon

a protein that activates gene expression upon binding DNA found specifically in E Coli

CRP-cAMP complex

a positive regulator of the lactose operon

A mutation in lacl does:

• not produces a functional repressor

• the lac genes are expressed in the presence and absence of lactose

One mutant and one non-mutant lacI does:

expressed lac genes only in the presence of lactose

A mutation in lacO does:

presents repressor protein from binding so the lac genes are expressed in the presence and absence of lactose and have constitutive expression.

One mutant and one non-mutant of lacO does:

lac genes are expressed in the presence and absence of lactose and have constitutive expression

Binds to operator

What do repressor proteins to?

lacI

• Produces a mRNA that encodes a protein called the lactose repressor protein

• Is outside of the lac operon

lacZ

Encodes enzymes needed to split lactose into smaller sugars

-produces B-galactosidase

lacY

encodes a membrane protein needed to transport lactose into the cell

-produces lactose permease

no repressor= transcription occurs

What happens when theres lactose present?

it is induced. The operator is not bound with the repressor, the promoter recruits the RNA polymerase complex and transcription of the polycistronic mRNA occurs.

in the presence of lactose, what happens to lactose operon?

Operon

the region of DNA consiting of the promoter, the operator, and the coding sequence for the genes

• found in bacteria/archae/ primarily in prokaryotes

• A set of multiple bacterial genes that are regulated together and are trasncribed into a single mRNA (they share a promoter)

• The part of the mRNA corresponding with each gene is translated into the protein encoded by that gene

• Usually contain multiple genes that functionally reglated proteins (they all contribute to the same biochemical/cellular process) and transcribed together

these cells can translate operon correctly due to their ribososomes ability to initiate translation anywhere along an mRNA that contains a proper ribosome-binding site.

Why do operon only found in bacteria and arcahe

Cistron

coding sequence

Polycistronic RNA

a single molecule of mRNA located next to one another along bacterial DNA

true

true or false: lacz and lac y are not transcribed together

Operator/ Lac O

the binding site for the repressor and its a sequence

between lacI and lacZ

Where do lacY, promoter, lacP and an operator located?

lacP

function is to recruit the RNA polymerase complex and initiate transcription

Structural gene

they ccode for the sequence of amino acids making up the primary structure of each protein

Trytophan

when sufficient, it binds with a regulatory protein to form the functional repressor, and the transcription of the genes does not occur

when drops too low, repressor does not form, transcription is initiated.

Positive Control

the abilitiy of the pressor to bind DNA OFTEN DETERMINED BY An allosteric interaction with a small molecule

Repressor

preventing transcription of the genes into messenger RNA.

Allosteric Effect

a change in the activity so the protein can bind to the molecule with the activator with the small molecule

5’ TATAA- 3’

helps recruit proteins needed for trasncription

negative regulation

must bind to the DNA at a site near the gene in order for transcription to be prevented.

• transcription takes place at a constant rate unless something turns it off

Transcriptional Regulation

the mechanisms that collectively regulate whenever transcription occus

• can be positive or negative

Positive Regulation

protein must bind to the DNA at a site near the gene in oorder for a transcription to take place

• Transcription is stimulated

DNA Sequence in

eukaryotes: Enhancer

Prokaryotes: Activator Sequence

Primary transcript

the initial RNA transcript of a gene is itself the mRNA immediately combines with ribosomes to undergo translation into protein

Gene Expression

the readout of genetic information flow in a cell that focuses on transcription, and translation that produce functional gene products.

Gene regulation

various ways in which cells can contol gene expression

• can occur at any steps, even after the protein is made

Eukaryotes vs Prokaryote gene expression

• In eukaryotes, it can be regulated at the level of chromosome packaging which involves the modification of histones, nucleosomes and DNA. They also have multiple enhancers

• in prokaryotes, it can be regulated at the level of transcription and environmental levels.

All cells

must be able to adjust which genes they re transcribing/translating at any given moment to respond in real time to environmental factors

Gene Regulation in Transcriptional Level

• Epigenetic Regulation (make DNA sequences more/less accessible for transcription)

• Binding of regulatory proteins to non-coding sequences on DNA (eg.TFs binding to enhancers in eukaryotes)

Eukaryotes

Where do gene regulation transcriptional level regulate only?

Gene Regulation Post Transcriptional Level

• How mRNA is processes

• Splicing

• mRNA stability

• only eukaryotes

Gene Regulation Translational Level

Whether initiation complex can assemble at 5’ cap

Negatively Regulated

Transcription is repressed

The organization of prokaryotic DNA is not packaged in chromatin, mRNA is not process, and no nuclear membrane seperating the processes of transcription and translation

Why is gene expression in general simpler than eukaryotes?

Metabolizing Glucose

provides greatest net energy “pay off”

E. coli can turn lactose into glucose and galactose

If glucose isn’t available:

Lac Operon

Purpose: to avoid wasting the energy and materials for making lactose metabolism proteins if they aren’t needed, E. coli can regulate the expression of the genes encoding these protein

• contains 3 genes that code for 3 proteins involved in lactose metabolism

Anatomy of Operon

DNA-operon starts at promoter-operator-structural genes(A-B-C-D) operon ends

lacz, lac, y , lac A

LacA

codes for another protein that lac operon contains

lacZ, lacY, lacA

What are the 3 codes that is involved in lactose metabolism?

lacP

Controls the transcription lacZ, lacY, lacA.

It eats it and break it down for energy

What do E-Coli do to lactose sugar?

E-Coli turns on genes that code for proteins

What happens if the lactose is present in the environment?

• E-coli keep these lactose metabolism gene off to avoid wasting energy.

• During these times, cells don’t need lactose metabolism proteins.

• LacI is constitutively continually and constantly expressed

If lactose is not present?

Lactose

Indirectly unrepresses lac operon transcription

Activating proteins factors

bind to genes’ enhancers

General transcription Factor

bind to the DNA at the promoter

Specific TF Proteinns

recognizze specific DNA sequences (enhancers and promoters)

Mediator Complex

• This is recruited when all TFs are bounded and then DNA loops

• Connects enhancers TFs and General Tfs and the promoter

Enhancers

influence on whether, where, when, and how much a promoters it attached to and how it’s going to be active

Promoter

• Acts like a switch on whether transcription would occurs or nah

• The sequence of DNA where RNA polymerase binds and begins transcription of the gene

Silencers

Represses transcription (negatively regulated)

Combinational Control

The different possible combination of various TFs bound to their respective regulatory regions for a gene provide multiple layers of fine-tuning, the timing, and speed of the gene’s transcription

The regulation of gene trasncription by means of multiple TFs acting together

Mutations

In regulatory, non-coding regions of DNA affects levels of transcription

Epigenetic Regulation

Changes in how DNA is packaged in the nucleus that affect gene transcription

• changes of bases

• chemical modification of histones

• chemical modificaiton of DNA

• the way in which DNA is packed within chromosomes/ alterations in chromatin structure / chromatin remodeling

only occurs in Eukaryotes

Chromatin

the complex of DNA and proteins that forms chromosomes. this is also The DNA that exists in a cell when it is not dividing is a thread-like structure formed by wrapping DNA around histones.

• Hereditary molecule wound around histones

Histones

in Eukaryotes, the proteins around which DNA is wound in each nucleosomes

Ways that a Eukaryotic Cells can control whether and how it uses a gene in the DNA to make a functional protein

• Controlling which transcriptions factors are available in the cell to bind to the genes regulatory sequences

• alternatively splicing the mRNA trasncript

• controlling the stability of the mRNA transcript

• blocking initiation factors from binding to the 5’ cap of the mRNA transcript

• modifying the protein product after translation to change its activity

B- Galactiosidase

the enzyme that breaks down lactose to galactose and glucose

Permease

Allow genes to enter the cell and facilitate transportation between the cell membrane organelles

Positive Regulation of the Lac Operon

A combination of unblocking and activating transcription

Whether and how much a gene is transcribed

depends on the exact combination of regulatory TFs and much they bound to their respective regulatory DNA sites

Alternative Splicing and RNA editing

How can one protein-coding gene code for more than one polypeptide chain

• through the action of small regulatory RNAs that can either inhibit translation of degrade mRNA

• Altering 5’ cap of the mRNA, so trnaslation initiation cannot occus

• altering or deleting the poly(A) tail, which inhibits translation

What are three ways in which gene expression can be influence after mRNA is processed and leaves the nucleus?

Regulatory Transcription Factors

• A protein that recruits the components of the transcription complex to the gene

• Highly diverse and specific to one or a subset of protein coding genes

• Mostly bind to enhancers

General Transcription Factors

Assemble near a short sequence in the promoted called the TATA box, the same set of protein for all protein-coding genes

RNA Processing

Primary transcript being modified several times and these modification include the 5’ cap and a string of about 250 adenosine nucleotides to the 3’ end to form the poly(A) tail

Poly A tail

helps to determine how long RNA will persist in the cytoplasm before being degraded. This also where translation is enhanced that bring the tail into contact w/ the initiation machinery on the 5’ Cap

Spliceosome

a biochemical machinery that exons are being in one during RNA splicings. The same sequences that are recognizd as exon can be recognized as intron in other RNA molecules.

RNA editing

the process in which some RNA molecules become a substrate for enzmes that modify particular bases in the RNA. Changings its sequence and what it codes for.

mRNA molecules

-they have 5’ cap, 5’ untranslated 5’ UTR, open reading frame (ORF), 3’ untranslated region, poly- A tail.

Open Reading Frame (ORF)

containing the codons that determine the amino acids/ sequences of the protein

5’ UTR

where proteins bind to transport mRNA to particular location in the cell or can affect translation initiation directly

3’ UTR

where translations are regulated

5’ and 3’ UTR

they contain regions that bind with regulatory proteins. These RNA binding proteins help control mRNA translation and degradation

Translation Initiation Complex

While some mRNA from a gene are being translated, others transcribed may not have these.

3’ UTR and Poly A Tail

initiation of translation

Post-translational Modification

Proteins are modified after translation in ways that regulate their structure and function. They help regulate activity and can alter protein folding and target molecules to particular cellular compartments.

Chaperones

Protein that help in proper folding

Different Level of Genes

Genes regulation can occur at any one or more of the steps of the gene expression: Chromatin, transcription, RNA processing, translation and post-translational modification.

Interphase and M-Phase

2 phases of cell cycles

Interphase

a process of growth and replication. Has 3 subphases. Moreover, during this, parent cell’s DNA must be faithfully replicated so that when the cell divides in two during M-phase, each daughter cell gets the exact genetic info.

G1, S1 and G2 phase

Interphase subphases

G1 Phase

-Increase volume of cytoplasm

-synthesizing new/more proteins

-producing more organelles

-building more plasma membrane to fit around all the stuff above

-Preparation of s-phase DNA synthesis

-in this checkpoint, p53 is involved

S Phase

entire DNA content in nucleus is replicated or where DNA synthesize. This is where DNA is being replicated

-this is when 46 total chromosomes gets replicated and cell divides in two, each new cell identical copies of these 2 sets chromosomes 1-23

G0

Phase

Cell that are not actively dividing

G2 Phase

• there are more growth and prepares for mitosis and cytokinesis (M-phase)

• Both the size and protein content increase in size

• in this checkpoint, it ensures DNA replication was successful and is complete

Complementary Daughter Strands

They are not synthesized the same way on both DNA templates (parents strands)

• are antiparallel to their respective parent templates

• their cells has a complete copy of DNA strand this means that the chromosome stays the same in mitosis

Leading Strand

The daughter strand with its 3’ end pointed towards replication fork, can be synthesized continuously

• they fray from 1 primer that is required to start synthesis

• Being added in the same direction as the replication fork opening