Chapter 13

Regulation of Gene Expression

The biochemistry and molecular biology department of CMU

Section 1

Principles and Concepts

§1.1 Concepts

Gene: A DNA segment that contains the all genetic information required to encodes RNA and protein molecules.

Genome: A complete set of genes of a given species.

Gene expression: A process of gene transcription and translation.

Specificity of gene expression

• Temporal specificity (also called stage specificity): why in the infant not in the aged ones?

• Spatial specificity (also called tissue specificity): why in liver not in brain?

Specificity of gene expression

Type of gene expression

a. Constitutive expression

Some genes are essential and necessary for life, and therefore are continuously expressed, such as those enzymes involved in TAC. These genes are called housekeeping genes.

b. Induction and repression

The expression levels of some genes fluctuate in response to the external signals.

Some genes demonstrate higher expression level once being activated. It is called induced expression.

On the other hand, some genes are repressed and their expression levels are lower. It is called repressed expression.

§1.2 Regulatory Elements

• Gene expression is a multiple-level process.

• Transcription initiation is a key point of controlling gene expression.

Basic elements that regulate the transcription include:

a. Special DNA sequences

b. Regulatory proteins

c. DNA-protein interaction and protein-protein interaction

d. RNA polymerase

For prokaryotic systems:

Operon is composed of structural genes, promoter, operator, and other regulatory sequences.

a. Special DNA sequence

Other requlatory sequence

Operator

Promoter Sturctural genes

The DNA sequence that RNA-pol can bind to and initiate the transcription.

Promoter

promoter operator structural generepressorRNA pol

The DNA sequence adjacent to the structural genes that the repressor protein can bind to and prevent the transcription of structural genes.

Operator

structural gene

start

AB

Cis-acting elements is the special DNA sequence that can affect the expression of its own gene.

For eukaryotic systems:

b. Regulatory proteins

For prokaryotic systems:

• Specific factor: It facilitates the binding of RNA-pol to particular DNA sequence.

• Repressor: It binds to the operator and prevent the transcription, known as negative regulation.

• Activator: It associates with DNA near the initiation point, resulting in the increase of RNA-pol binding affinity and the enhancement of the transcription efficiency.

For eukaryotic systems:

• The regulatory proteins are called transcription factors (TF).

• After expression, TF will interact with the cis-acting elements to activate another genes. Therefore, they are referred to as trans-acting factors.

Trans-acting factors

a

A

DNA

mRNA

protein A

bA

• The regulation is implemented through numerous interactions between cis-acting elements and trans-acting factors.

• They are non-covalent bond.

c. DNA-protein interactions

• Proteins may have to interact with each other prior to the DNA binding.

• Proteins can form a homo or hetero-dimer form to function properly.

• Present in prokaryotes as well as eukaryotes.

Protein-protein interactions

Section 2

Gene Regulation of Prokaryotic Systems

Common features

• Prokaryotic genes are polycistron systems, that is, several relevant genes are organized together to form a transcription unit --- operon.

• The majority of gene regulation is negative. Inducers are used to remove the repression.

• Operon is a coordinate unit for the reg

ulation.

• Transcription initiation is the key point for regulation. Translation can also be regulated.

§2.1 Regulation of Transcription

AYZOPI

structural gene

permease

¦Â-galactosidase

regulatory site

operator

promoter

CAP-binding site

regulatory gene

transacetylase

Structure of lac operon

Metabolism of lactose

• Bacteria do not express these three enzymes when glucose is available. However, bacteria produce those enzymes if lactose is present and glucose is absent.

Inducible expression

Sequence of lac operon

• lac operon (TTTACA/TATGTT) is a weak promoter, and has a basal expression level.

• CAP (Catabolite gene activator protein) binding site is at -60 region.

• CAP is a homodimer with binding ability to DNA and cAMP.

• Glucose inhibits the formation of cAMP.

• When glucose is present, [cAMP] is lower. Only after glucose is exhausted, [cAMP] becomes higher. The CAP-cAMP complex is formed, and this complex binds to the CAP binding site on lac operon.

AYZOPI

mRNA

RNA pol

When lactose is absent, no lac gene is expressed.

Situation 1

• lacI gene has its own promoter, and its expression can produce LacI repressor.

• The tetrameric Lac repressor binds to the lac operator site Olac.

• The binding blocks the RNA-pol moving on DNA template, and no lacZ, lacY, and lacA are expressed.

AYZOPI

repressor

mRNA

lactoseallolactose

RNA pol

galactosidase

Situation 2

When lactose is present, lacZ, lacY, and lacA genes are expressed.

• The galactosidase is weakly expressed (at the basal level).

• When lactose is present, it is converted to allolactose or galactose that binds to the repressor.

• The repressor can no longer bind to the operator, and lac gene can be expressed.

• Galactose , Allolastose and IPTG are referred to as inducer.

Inducers

• The lacZYA RNA transcript is very unst

able and could be degraded quickly. Therefore, the synthesis of three enzymes will be cease under normal condition.

Presence of lactose

AYZOPI RNA pol

CAP

When glucose is present, the [cAMP] is low, no CAP-cAMP is formed and the expression of the lac operon is still low.

Situation 3

AYZOPI

CAP

cAMP

RNA pol

When glucose is absent and lactose is present, the CAP-cAMP complex binds to the CAP site to activate the lac gene.

Situation 4

O O

OO

RNA pol

lactoseno lactose

RNA pol

RNA pol RNA pol

Coordinate expression

No glucose

Glucose

§2.2 Transcription Attenuation

• The trp operon is one of the constitutive genes expressed at the basal level.

• The structural gene of trp operon encodes 5 enzymes used for the synthesis of Trp.

Trp operon

• The trp repressor gene can be expres

sed, but it does not bind to the operator.

• When Trp is more than enough, the repressor will form a complex with Trp. The complex binds to the operator, blocking the synthesis of Trp.

ABCDEOPR

regulatory regions

structuralgene

Trp

Trp mRNA

attenuated mRNA

trp L attenuator

Trp operon

Attenuation mechanism

• In addition to the repressor regulation, trp gene has a fine tuning mechanism called attenuation.

• The trp operon is regulated using attenuation mechanism at the translation level.

Leader sequence

Possible hairpins

1/2 and 3/4 hairpin

structure2/3 hairpinstructure

High Trp concentration

Low Trp concentration

• Under the normal conditions, the Lex

A gene expressed to repressor proteins that bind to promoters of other genes and block their expressions.

• Once the repressors are degraded, the repressed genes will be expressed.

• At the basal level, the normal cell contains about 1000 copies of RecA protein.

§2.3 Protein Degradation

SOS response

LexA digestion

• When DNA is extensively damaged, DNA replication is halted and the number of ssDNA gaps increases.

• The RecA protein binds to this damaged ssDNA, which activates the protein’s coprotease activity.

• While bound to ssDNA, the RecA protein facilitates the cleavage of LexA repressor as well as the inactivation of the LexA repressor.

P1 H1P2hin H2 repressor

DNA

H2 flagellinHin recombinase

P1 H1

P2 hin

H2 repressor

H1 flagellintransposed segment

§2.4 Genetic Recombination

• An RNA, with sequence

complementary to a specific RNA

transcript or mRNA, whose binding

prevents processing of the transcript

or translation of the mRNA.

Antisense RNA

Antisense RNA

Section 3

Regulation of Eukaryotic Transcription

Structural features

• Large genome: 3 x 109 bps, 35 000 genes

• Monocistron

• Repeated sequences: different lengths and different frequencies. Often inverted repeats

• Splite genes: separated by introns and exons alternatively

Regulation features

1. RNA-pol: 3 forms (I, II, and III) for different RNAs

2. Changes of chromosomal structure • Hypersensitive site

• Base modification

• Isomer-conversion

• Histone changes

3. Positive regulation

4. Transcription and translation are separated

5. Post-transcriptional modification

6. Regulation through intercellular and intracellular signals

§3.1 Cis-acting elements

• They are specific DNA sequences, each of which regulates transcription of one or more genes. They usually have consensus sequences.

• Promoter: TATA box, CAAT box, and GC box,

• Sequence: TATAAAA

• Location: - 25 ~ - 30 bp

• Function: It is the binding site for

TFII D, which is required for RNA pol

ymerase binding. It controls the ver

acity and frequency of transcription

al initiation.

TATA box

• Sequence: GCCAAT

• Location: ~ -70 bp

• Function: It is the binding site for C

TF1 (CAAT-binding transcription fact

or) and C/EBP (enhancer binding pro

tein).

CAAT box

• Sequence: GGGCGG

• Location: -30 ~ -110 bp

• Function: It is the binding site for a protein called Sp1.

GC box

• It is a DNA sequence that can determine the temporal and spatial specificities of expression and increase the promoter activity.

enhancer

• It is a negative regulation element.

• It will repress the transcription once interacted with specific proteins.

Silencer

§3.2. Trans-acting factors

• They are the proteins that bind indirectly to cis-acting elements and then regulate the transcription initiation.

• The trans-acting factors can be transcription factors (TF).

transcription factors

• General transcription factors

• Special transcription factors– Transcription activators

EBP (enhancer binding protein)

– Transcription inhibitors

General structure of TF

• DNA-binding domain

• Activation domain

• Protein-protein interaction domain

Promoter and regulatory proteins

General structure of TF

• CTD of RNA-pol II is an important point of interaction with mediators and other protein complexes.

• Cofactors facilitate the TF assembly.

Transcription repressor

§3.3 DNA-protein interactions

• Regulatory proteins have discrete DNA-binding domains of particular structure, i.e., binding motif.

• The AA side chains of regulatory proteins interact with bases of DNA through H bonds.

Yeast activator protein GCN4

Leucine zipper

Zinc finger

Steroid hormone receptor

Mouse regulatory protein Zif268

Zinc finger

Helix-loop-helix

Helix-loop-helix

Human transcription factor MAX

Helix-turn-helix

Lac repressor

Helix-turn-helix

Trp repressor