PRINCIPLES OF

MOLECULAR
BIOLOGY-SEMINAR
B Y,
IDA NANCY K
I II YE AR
I NT.MSC . LI FE SCIE NC E S
TRANSCRIPTIONAL
REGULATION
ACTIVATION – STAT PROTEINS; ACTIVATION OF
TRANSCRIPTIONAL ELONGATION BY HIV TAT PROTEINS
 TRANSCRIPTION FACTORS
•Transcription factors are proteins that help turn specific genes "on" or "off" by binding to
nearby DNA.

•Transcription factors that are activators boost a gene's transcription. Repressors decrease

transcription.

•Groups of transcription factor binding sites called enhancers and silencers can turn a gene

on/off in specific parts of the body.

•Transcription factors allow cells to perform logic operations and combine different sources of
information to "decide" whether to express a gene.
REGULATION BY PHOSPHORYLATION-
STAT PROTEINS
Certain hormones bind to cell-surface receptors and pass a signal to proteins

within the cell through signal transduction.

In a signal transduction pathway, a specific ligand binds to an extracellular

domain of a specific cell surface receptor and this binding brings about an

allosteric change in the intracellular domain of receptor leading to activation of

a series of protein kinases ultimately resulting in the activation of a transcription

factor.

Signal transduction often involves protein phosphorylation.

EXAMPLE
Example: Interferon-γ induces phosphorylation of a transcription factor called

STAT1α through activation of the intracellular kinase called Janus activated

kinase(JAK).
STAT PROTEINS
oMembers of the signal transducer and activator of transcription (STAT) protein family are
intracellular transcription factors that mediate many aspects of cellular immunity, proliferation,
apoptosis and differentiation.

oThey are primarily activated by membrane receptor-associated Janus kinases (JAK).

oDysregulation of this pathway is frequently observed in primary tumors and leads to increased
angiogenesis which enhances the survival of tumors and immunosuppression.
1. Unphosphorylated STAT1α protein: exists as a monomer in the

cell cytoplasm and has no transcriptional activity.

2. Phosphorylated STAT1α at a specific tyrosine residue forms a

homodimer which moves into the nucleus to activate the

expression of target genes whose promoter regions contain a

consensus DNA-binding motif

JAK STAT PATHWAY
oThe JAK-STAT signaling pathway is a chain of interactions between proteins in a cell, and is
involved in processes such as immunity, cell division, cell death and tumour formation.

oThe pathway communicates information from chemical signals outside of a cell to the cell
nucleus, resulting in the activation of genes through a process called transcription.

oThere are three key parts of JAK-STAT signalling: Janus kinases (JAKs), signal transducer and
activator of transcription proteins (STATs), and receptors (which bind the chemical signals)
MECAHNISM
oThe binding of various ligands, usually cytokines, such as interferons and interleukins, to cell-
surface receptors, causes the receptors to dimerize, which brings the receptor-associated JAKs
into close proximity.

oThe JAKs then phosphorylate each other on tyrosine residues located in regions called activation
loops, through a process called transphosphorylation, which increases the activity of their kinase
domains.

oThe activated JAKs then phosphorylate tyrosine residues on the receptor, creating binding sites
for proteins possessing SH2 domains
oSTATs then bind to the phosphorylated tyrosines on the receptor using their SH2 domains, and
then they are tyrosine-phosphorylated by JAKs, causing the STATs to dissociate from the
receptor.

oAt least STAT5 requires glycosylation at Threonine 92 for strong STAT5 tyrosine
phosphorylation
oThese activated STATs form hetero- or homodimers, where the SH2 domain of each STAT binds
the phosphorylated tyrosine of the opposite STAT, and the dimer then translocates to the cell
nucleus to induce transcription of target genes.

oSTATs may also be tyrosine-phosphorylated directly by receptor tyrosine kinases - but since
most receptors lack built-in kinase activity, JAKs are usually required for signalling
MOVEMENT OF STAT PROTEINS
FROM CYTOSOL TO NUCLEUS
oTo move from the cytosol to the nucleus, STAT dimers have to pass through nuclear pore
complexes (NPCs), which are protein complexes present along the nuclear envelope that control
the flow of substances in and out of the nucleus.

oTo enable STATs to move into the nucleus, an amino acid sequence on STATs, called the nuclear
localization signal (NLS), is bound by proteins called importins
oOnce the STAT dimer (bound to importins) enters the nucleus, a protein called Ran (associated
with GTP) binds to the importins, releasing them from the STAT dimer.[8] The STAT dimer is
then free in the nucleus.
HOW STAT PROTEINS
ACTIVATE TRANSCRIPTION?
oOnce STAT reaches the nucleus, it binds to a consensus DNA-recognition motif called gamma-
activated sites (GAS) in the promoter region of cytokine-inducible genes and activates
transcription.

oThe STAT protein can be dephosphorylated by nuclear phosphatases, which leads to inactivation
of STAT and subsequent transport out of the nucleus by an exportin-RanGTP complex.
WHAT DO STAT PROTEINS
TRANSCRIBE ?
oSTATs help promote the survival and function of the cytotoxic (killer) T-cells and NK-cells.

oCytokines – these are proteins that are produced by immune cells during an immune response.
Some cause inflammation (pro-inflammatory) while others suppress inflammation (anti-
inflammatory).

oSurvival proteins – these proteins perform functions in the cell that allow the cells to stay alive.
TRANCRIPTIONAL ELONGATION
ACTIVATION BY HIV TAT PROTEINS
Human immunodeficiency virus (HIV) encodes an transcriptional

activator protein called Tat, which is required for productive HIV

gene expression.

Tat binds to an RNA stem-loop structure called TAR, which is

present in the 5’-UTR of all HIV RNAs just after the HIV transcription

start site, to regulate the level of transcription elongation.

HIV TAT PROTEINS
oIn molecular biology, Tat is a protein that is encoded for by the tat gene in HIV-1.

oTat is a regulatory protein that drastically enhances the efficiency of viral transcription.

oTat stands for "Trans-Activator of Transcription".

oThe protein consists of between 86 and 101 amino acids depending on the subtype.

oTat vastly increases the level of transcription of the HIV dsDNA.

FUNCTION AND MECHANISM
oLike other lentiviruses, HIV-1 encodes a trans-activating regulatory protein (Tat), which is
essential for efficient transcription of the viral genome.

oTat acts by binding to an RNA stem-loop structure, the trans-activating response element (TAR),
found at the 5′ ends of nascent HIV-1 transcripts.

oIn binding to TAR, Tat alters the properties of the transcription complex, recruits the positive
transcription elongation complex (P-TEFb) of cellular CDK9 and cyclin T1, and hence increases
the production of full-length viral RNA
oTat protein also associates with RNA polymerase II complexes during early transcription
elongation after the promoter clearance and before the synthesis of full-length TAR RNA
transcript.

oThis interaction of Tat with RNA polymerase II elongation complexes is P-TEFb-independent.

There are two Tat binding sites on each transcription elongation complex; one is located on TAR
RNA and the other one on RNA polymerase II near the exit site for nascent mRNA transcripts
which suggests that two Tat molecules are involved in performing various functions during a
single round of HIV-1 mRNA synthesis
 In the absence of Tat, the HIV transcripts terminate prematurely due

to poor processivity of the RNA Pol transcription complex.

Tat binds to TAR on one transcript in a complex together with

cellular RNA-binding factors. This protein-RNA complex may loop

backwards and interact with the new transcription initiation complex

which is assembled at the promoter.

This interaction results in the activation of the kinase activity of TFIIH,

leading to phosphorylation of the carboxyl-terminal domain (CTD) of

RNA Pol, making the polymerase a processive enzyme to read

through the HIV transcription unit, leading to the productive

synthesis of HIV proteins.