Gen Ekspresi

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.

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

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

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

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.

The regulation is implemented through numerous interactions between cis-acting elements and trans- acting factors. They are non-covalent bond. 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

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

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.

Regulation of Eukaryotic Transcription

Structural features Large genome: 3 x 10 9 bps, 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

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 polymerase binding. It controls the veracity and frequency of transcriptional initiation. TATA box

Sequence: GCCAAT Location: ~ -70 bp Function: It is the binding site for CTF1 (CAAT-binding transcription factor) and C/EBP (enhancer binding protein). 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

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

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

DNA sequencing Determination of nucleotide sequence  the determination of the precise sequence of nucleotides in a sample of DNA Two similar methods: 1. Maxam and Gilbert method 2. Sanger method They depend on the production of a mixture of oligonucleotides labeled either radioactively or fluorescein, with one common end and differing in length by a single nucleotide at the other end This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrilamide gels and the position of the bands determined

CyclophosphamideCytoxan® alkylating agents; form interstrand and/or intrastrand crosslinks MelphalanAlkeran® BusulfanMyleran® ChlorambucilLeukeran® MitomycinMutamycin® CisplatinPlatinol®forms crosslinks BleomycinBlenoxane®cuts DNA strands between GT or GC IrinotecanCamptosar® inhibit the proper functioning of enzymes (topoisomerases) needed to unwind DNA for replication and transcription MitoxantroneNovantrone® DactinomycinCosmegen®inserts into the double helix preventing its unwinding The table lists (by trade name as well as generic name) some of the anticancer drugs that specifically target DNA.