AGR Exam 2

Levels of gene regulation: Transcriptional ______________

initiation

What do structural genes do?

encode proteins that play essential roles in metabolism, biosynthesis, or other critical structural role in cells

What do regulatory genes do?

genes whose products, either RNA or protein, interact with DNA sequences of other structural genes and affect their transcription or translation.

➢ Many are DNA binding proteins - regulatory proteins

What are regulatory elements?

DNA sequences, where the proteins produced by regulatory genes recognize and bind.

What are two processes of gene expression regulation?

Stimulate gene expression: positive control

Inhibit gene expression: negative control

DNA binding proteins

regulatory proteins that bind to regulatory element of DNA sequence and affect gene expression at transcriptional level.

Where are DNA binding proteins at and responsible for?

Discrete functional parts → Domains, 60-90aa, responsible for DNA binding

What are the motifs for DNA Binding proteins?

a few aa within the domain, that can form hydrogen bonds with DNA nucleotides (either base or sugar backbone)

Are DNA binding proteins fixed or not?

Binding is dynamic not permanent (can be active or inactive)

How do bacterial gene with related functions cluster or not?

Bacterial genes with related functions are often clustered together with one single promoter and transcribed together into a single mRNA

Definition of operon

a group of bacterial functional genes that are transcribed together along with the controlling sequences.

How are opreons organized?

Promoter-Initiates the structural gene expression

Operator-DNA sequence bound by regulatory protein to regulate transcription

between the promoter and the structural genes

Structural genes- encodes functional proteins

Regulatory gene of an operon is a separate gene - encodes regulator protein with its own promoter. Can be active or inactive.

Where is the operator typically located?

The operator is a DNA sequence usually at the border between the promoter and the structural genes.

What are the two types of transcriptional control?

Negative control and positive control.

What does the regulator protein do under negative control and positive?

Negative: Repressor
Binds to DNA to block/inhibit

Positive: Activator

Binds to DNA to stimulate transcription

Negative inducible Operon

Normally, the regulatory protein (a repressor) is active and bound to the operator to prevent operon transcription. So, an active repressor binds to operator = no transcription

For the negative inducible operon, what happens if a precursor (inducer) is present what happens?

If the precursor (inducer) is present, the repressor can’t bind = transcription on

Negative repressible operons

Normally transcription of the operon is on. Regulator protein = repressor. The repressor is typically inactive
Inactive repressor = no binding on operator = transcription is on

How do the negative repressible operons function/operate with a corepressor?

If repressed by corepressor, a small molecular (product), which can bind to the inactive repressor to activate it. Activated repressor bind to the operator. Transcription stops. Product (co-repressor) present = repressor binding = no transcription.

Negative control → regulatory protein = ?

represses transcription

Positive control → regulatory protein = ?

activator, stimulates transcription

Inducible

normally structural genes are not transcribed. Inducers (substrate or precursor) can induce the transcription (by activate activator or inactivate repressor)

Repressible

Normally structural genes are transcribed. Corepressors (products) can repress the transcription (by inactivate activator or activate repressor)

Positive inducible operons

Transcription normally is off. The regulator protein (activator) can be activated by inducer to bind to DNA (a site other than the operator) and to activate transcription

Positive repressible operons

Normally, the regulatory protein (activator) binds to DNA and activates transcription. Product (corepressor) inactivates the activator and transcription turns off.

lac operon

controls metabolism of the lactose in cell

lacl

regulator gene with its own promoter → encode a repressor

lacP

common promoter for 3 structural genes

lacO

operator where the repressor binds

what are the 3 adjacent structures for the lac operon?

lacZ, lacY, lacA. Z encodes a B-galactosidase, which converts lactose into glucose, Y encodes permease allowing lactose to enter the cell, A encodes transacetylase with unknown function.

What type of operon is the lac operon?

When lactose or allolactose (substrate) is present in the medium, where the bacteria grows, all 3 genes are expressed to metabolize lactose → inducible
An example of negative inducible operon

Positive control for the lac operon?

Lac operon show positive regulation when glucose is present (product, as a repressor) is present at high levels, regardless if lactose is present or not, lac operon will be turned off. Results in efficient energy use in cell.

When glucose level increases, what does the CAP mechanism control?

CAP mechanism controls the transcription of the lac operon, no matter what the lactose level is.

What type of control is the CAP associated with?

Positive control. CAP binds to cyclic adenosine monophosphate (cAMP) → cAMP-CAP = activator

What purpose does the cAMP serve?

cAMP-CAO binds to CAP site and interacts with RNA polymerase to stimulate lac operon transcription. Glucose level regulates cAMP level. High glucose = low cAMp Low glucose = high cAMP

trp operon

produces enzymes to synthesize tryptophan

What happens in low tryptophan, when tryptophan is present, and what type of system is it?

Regulator gene (trpR)
➢ Product = regulator protein → repressor
Low tryptophan levels
➢ Repressor is inactive and can’t bind to DNA operator
➢ RNA polymerase binds DNA → transcription on
✓ tryptophan is synthesized
Tryptophan present
➢ It serves as a corepressor to activate the repressor
➢ Activated repressor then binds to DNA operator
➢ Inhibiting RNA polymerase binding → no transcription
An example of negative repressible gene system

In eukayotes, what are the structure of chromosomes?

Chromosomes are tremendously packed and condensed and consist of organized DNA.

DNA + histones = ?

NUcleosomes

what consist of a histone octamer?

H2A, H2B, H3, H4

Nucleosomes fold up and compress into what?

chromatin fiber. it can also be further condensed, chromatin fiber coiled into a chromatid and chromatids constitute a chromosome.

Euchromatin

refers to the uncondensed chromosome regions.
➢ Constitutes the majority of chromosome material.
➢ Actively transcribed genic regions

Heterochromatin

condensed chromosome regions
➢ Contains mostly non-coding DNA (lack of genes).
➢ Centromere and telomere regions (Lecture 13).
➢ Long sequences of tandem repeats

How is eukaryotic gene expression more complex in eukaryotes than in prokaryotes?

Packaged as chromatin (DNA + proteins)
➢ Condensed (closed) or uncondensed (________) chromatin = On/off switch for regulation
Processing of pre-mRNA to mature mRNA
mRNA half-life
➢ Prokaryotic – decays in minutes
➢ Eukaryotic – much longer
Site of transcription is separated from translation

Chromatin Structure Alteration for Regulation

Generally, chromatin structure represses/inhibits expression gene
➢ Condensed, coiled, inaccessible to transcription factors and RNA polymerases
• Prior to transcription, chromatin structure altered to open and expose the promoter region.
➢ E.g. Modification of histone proteins: methylation and acetylation

What is the purpose of DNA methylation?

Methylated DNA is associated with the repression/silencing of transcription. Histone methylation or acetylation mostly activates gene expression. Most common on cytosine adjacent to guanine.

GAL4

GAL4 is a transcriptional activator protein
• Regulates transcription of genes, whose products metabolize galactose
• Binds to UASG enhancer
• No galactose, GAL80 binds to GAL4 → no transcription
• When galactose is present GAL80 can’t bind to GAL4, GAL4 →activates transcription

Gene regulation through splicing

Alternative splicing determines sexual development in Drosophila
• Sxlgene regulates the splicing of Tra (transformer) gene
• In XX embryos, the Sxl is activated and cause Tra properly processed.
• Tra and Tra-2 direct the female-specific splicing of Dsx pre-mRNA, which cause embryo to develop into a female.

RNA interference for gene regulation

RNA interference (RNAi), also known as RNA silencing and post-transcriptional gene silencing.
• 30% human genes are negatively regulated by RNAi
• Triggered by microRNAs (miRNAs) and small interfering RNAs (siRNAs)
➢ About 22 nt small RNA ➢ Generated from double-stranded RNA
➢ Differ in how they originated

What are three mechanisms of RNAi for gene regulation?

Cleavage of mRNA ➢ RISC: RNA induced silencing complex. A single strand miRNA or siRNA pairs with protein to form the RISC
➢ siRNA in RISCs pairs with mRNA and the RISC cleaves mRNA
➢ After cleavage, the mRNA is further degraded.
➢ mRNAs are broken down and the amount of protein is decreased

• Inhibition of translation
➢ miRNA in RISCs matches the mRNA imperfectly
➢ inhibits translation of the targeted mRNA

• Transcriptional silencing ➢ Some siRNAs combine with proteins and form a complex called RITS (RNA induced transcriptional silencing)
➢ siRNAin RITS binds to DNA and causes methylation and restricting transcription