final summer internship report new_

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Analysis of Transcriptional

Interference in Gene Regulation

By

Panchanan Verma

SRFP 2015 Application No. LFS752

Summer Research Fellowship Programme 2015

Conducted by Indian Academy of Sciences, Bangalore

Guide: - Dr. Supreet Saini

Indian Institute of Technology, Bombay, Mumbai

Department of Chemistry

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Table of Contents

1- Project Title 2- Description A) Gene Expression B) Regulation Of gene expression C) Prokaryotic Transcription D) RNA Polymerase

E) Transcriptional Interference

3- Analysis of Transcriptional Interference in

gene regulation A) Based on strength of promoters B) Table and Graph between rate of Transcription and Transcriptional

Interference (%) on M. EXCEL.

4-Dynamics of Rate of Transcription

5-Random Stimulation bases on Probability

Distribution on MATLAB.

6-Conclusion

SUMMER INTERNSHIP REPORT

Project Title: - Analysis of T

Interference in Gene R

Description: gene's information is converted into the structures and functions of

a cell by a process of producing a biologically functional molecule

of either protein or RNA (gene product) is made

The process of gene expression is used by all known life

eukaryotes (including multicellular organisms), prokaryotes

(bacteria and archaea), and ut

macromolecular machinery for life

Several steps in the gene expression process may be modulated,

including the transcription

translational modification

Gene expression is assumed to be controlled at various points in the

sequence leading to protein

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Analysis of Transcriptional

Interference in Gene Regulation.

- Gene expression is the process by which a

gene's information is converted into the structures and functions of

a process of producing a biologically functional molecule

of either protein or RNA (gene product) is made.

The process of gene expression is used by all known life


(bacteria and archaea), and utilized by viruses - to generate the

macromolecular machinery for life.


transcription, RNA splicing, translation

modification of a protein.

is assumed to be controlled at various points in the

sequence leading to protein synthesis.


ranscriptional

he process by which a

gene's information is converted into the structures and functions of

a process of producing a biologically functional molecule

The process of gene expression is used by all known life -


to generate the


translation, and post-

is assumed to be controlled at various points in the

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Some simple examples of where gene expression is

important are:

Control of insulin expression so it gives a signal for blood glucose regulation.

X chromosome inactivation in female mammals to prevent an "overdose" of the genes it contains.

Cyclin expression levels control progression through the eukaryotic cell cycle.

Regulation of gene expression: - The first system of gene

regulation that was understood was the Lac Operon in E. coli, worked out by

Francois Jacob and Jacques Monod in 1962.

Regulation of gene expression includes a wide range of mechanisms

that are used by cells to increase or decrease the production of

specific gene products (protein or RNA), and is informally termed

Gene Regulation.

Gene regulation gives the cell control over structure and function, and

is the basis for cellular differentiation, morphogenesis and the

versatility and adaptability of any organism. Gene regulation may also

serve as a substrate for evolutionary change, since control of the

timing, location, and amount of gene expression can have a profound

effect on the functions (actions) of the gene in a cell or in a multicellular

organism.

Regulated s

Any step of gene expression may be modulated, from the DNA

RNA transcription

protein. The following is a list of stages where gene expression is

regulated; the most extensively utilized point is Transcription

Initiation:

Chromatin domains

Transcription

Post-transcriptional modification

RNA transport

Translation mRNA degradation

Gene expression must be regulated in several different

dimensions—

In Time:

At different stages of the life cycle, different genes need to be

on and off.

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Regulated stages of gene expression

Any step of gene expression may be modulated, from the DNA

RNA transcription step to post-translational modification of a



Chromatin domains

Transcription

transcriptional modification

RNA transport

Translation mRNA degradation



tages of gene expression

Any step of gene expression may be modulated, from the DNA-

translational modification of a





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Examples of gene regulation Enzyme induction is a process in which a molecule (e.g., a drug) induces

(i.e., initiates or enhances) the expression of an enzyme. The induction of heat shock proteins in the fruit fly Drosophila melanogaster. The Lac operon is an interesting example of how gene expression can be

regulated. Viruses, despite having only a few genes, possess mechanisms to regulate

their gene expression, typically into an early and late phase, using collinear systems regulated by anti-terminators (lambda phage) or splicing modulators (HIV).

GAL4 is a transcriptional activator that controls the expression of GAL1, GAL7, and GAL10.

• Negative and Positive Regulation: - The lac operon is negatively regulated: the regulatory protein (repressor) causes transcription to stop. Positive regulation, where the regulatory protein causes transcription to start, is more common. The lac operon also contains an example of positive regulation, called “catabolite repression”.

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Mechanisms of gene regulation include:

Regulating the rate of transcription. This is the most economical method of regulation.

Regulating the processing of RNA molecules, including alternative splicing to produce more than one protein product from a single gene.

Regulating the stability of mRNA molecules.

Regulating the rate of translation.

1. Transcription control of gene regulation is controlled by:

1. Promoters

• Occur upstream of the transcription start site.

• Some determine where transcription begins (e.g., TATA), whereas others

determine if transcription begins.

• Promoters are activated by specialized transcription factor (TF) proteins (specific

TFs bind specific promoters).

• One or many promoters (each with specific TF proteins) may occur for any given

gene.

• Promoters may be positively or negatively regulated.

2. Enhancers

• Occur upstream or downstream of the transcription start site.

• Regulatory proteins bind specific enhancer sequences; binding is determined by

the DNA sequence.

• Loops may form in DNA bound to TFs and make contact with upstream enhancer

elements.

• Interactions of regulatory proteins determine if transcription is activated or

repressed (positively or negatively regulated).

Control of Gene Expression

Lac Operon: - The Lac operon acts like a switch

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Control of Gene Expression: -

operon acts like a switch.

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Prokaryotic Transcription: - It is the process in which mRNA transcripts of

genetic materials in prokaryotes are produced, to be translated for production of

proteins. It occurs in the cytoplasm alongside translation.

Several different types of RNA are produced, including messenger RNA(mRNA),

which specifies the sequence of amino acids in the protein product, plus transfer

RNA (tRNA) and ribosomal RNA (rRNA), which play a role in the translation

process.

There are Four distinct stages of transcription:-

1) Initiation, 2) Elongation, 3) Termination 4) Processing

1) Initiation: - The regions of the DNA that signal initiation of transcription in

prokaryotes are termed promoters. (A)RNA polymerase searches for a promoter site. (B) It recognizes a promoter site and binds tightly, forming a closed complex. (C) The holoenzyme unwinds a short stretch of

DNA, forming an open complex. Transcription begins, and the σ factor is released. The synthesis of RNA proceeds in a 5' to 3' direction, so the template strand must be 3' to 5'.

2) Elongation: - Shortly after initiating transcription, RNA polymerase moves along the template strand, synthesizing an mRNA molecule. the sigma factor dissociates from the RNA polymerase. The RNA is always synthesized in the 5′ → 3′ direction with nucleoside triphosphates (NTPs) acting as substrates for the enzyme.

3) Termination: - Two termination mechanisms are well known:

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Intrinsic termination (also called Rho-independent transcription termination) involves terminator sequences within the RNA that signal the RNA polymerase to stop. The terminator sequence is usually a palindromic sequence that forms a stem-loop hairpin structure that leads to the dissociation of the RNAP from the DNA template. Rho-dependent termination uses a termination factor called ρ factor (rho factor) which is a protein to stop RNA synthesis at specific sites. This protein binds at a rho utilization site on the nascent RNA strand and runs along the mRNA towards the RNAP.

4) Processing: - After transcription the RNA molecule is processed in a number of ways: introns are removed and the exons are spliced together to form a mature mRNA molecule consisting of a single protein-coding sequence. RNA synthesis involves the normal base pairing rules, but the base thymine is replaced with the base uracil. Transcription factors are proteins that play a role in regulating the transcription of genes by binding to specific regulatory nucleotide sequences. Transcription Enzyme - RNA polymerase

RNA polymerase

(RNAP) is an enzyme that produces primary transcript RNA. In cells, RNAP is

necessary for constructing RNA chains using DNA genes as templates, a

process called transcription. RNA polymerase enzymes are essential to life and

are found in all organisms and many viruses. In chemical terms, RNAP is a

nucleotidyl transferase that polymerizes ribonucleotide at the 3' end of an RNA

transcript.

The core enzyme contains two α polypeptides, one β polypeptide, and one β′

polypeptide. The beta (β) subunit has a molecular weight of 150,000 , beta

prime (β′) 160,000, alpha (α) 40,000, and sigma (σ) 70,000.The σ subunit can

dissociate from the rest of the complex,leaving the core enzyme. The complete

enzyme with σ is termed the RNA polymerase Holoenzyme and is necessary

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for correct initiation of transcription, whereas the core enzyme can continue

transcription after initiation.

Structure of RNA polymerase: -

This enzyme that controls transcription and is characterized by:

Search DNA for initiation site,

It unwinds a short stretch of double helical DNA to produce a single-stranded DNA

template,

It selects the correct ribonucleotide and catalyzes the formation of a phosphodiester

bond,

It detects termination signals where transcript ends.

Eukaryotic RNA polymerases have different roles in transcription.

Polymerase I Nucleolus Makes a large precursor to the major rRNA (5.8S,18S and 28S rRNA in vertebrates

Polymerase II Nucleoplasm Synthesizes hnRNAs, which are precursors to mRNAs. It also make most small nuclear RNAs (smRNAs)

Polymerase III Nucleoplasm Makes the precursor to 5SrRNA, the tRNAs and several other small cellular and viral RNAs.

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Transcriptional Interference

Transcriptional Interference usually refers to the direct negative impact of one

transcriptional activity on a second transcriptional activity in cis. Transcriptional

interference (TI) specifically as the suppressive influence of one transcriptional

process, directly and in cis on a second transcriptional process.

TI can arise as a result of several different promoter arrangements: -

(a) Convergent promoters, directing converging transcripts that overlap for at least part of their sequence,such as the coliphage 186 lytic and lysogenic promoters;

(b) Tandem promoters, one upstream of the other but transcribing in the same direction, with their transcripts possibly but not necessarily overlapping, for example, the promoters of yeast SRG1 and SER3;

(c) Overlapping promoters- either divergent, convergent or tandem, in which the two RNAP-binding sites share at least a common DNA sequence, such as the E. coli aroP P1 and P3 promoters.

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Mechanisms of Transcriptional Interference: -Of the mechanisms of

TI that we will outline, three are implemented at the initiation phase of transcription and

two at the elongation stage.

Mechanisms of transcriptional interference (TI). Five possible mechanisms by which TI

can occur are: (a) promoter competition; (b) sitting duck interference; (c) occlusion; (d)

collision; and (e) roadblock. For the example shown here where a strong (aggressive)

promoter pA is oriented convergently to a weak (sensitive) promoter pS, all five

mechanisms are possible. For promoters arranged in tandem, all mechanisms except the

collision mechanism [shown in (d)] can apply. When the promoters are arranged

divergently, only the promoter competition mechanism shown in (a) can apply, but only

when the promoters are also overlapping.

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Transcriptional Interference: - There are three cases arises when RNAP collision occurred:-

Suppose - Two promoters P (L) and P(R) are moving along opposite side

on the axis depending on the different strength of promoters.

CASE 1= When there is Equal strength of promoters on both LHS and RHS

sides i.e. (P (L) = P(R) = 1RNAP / X- b-base).

Suppose if P(L)=P(R)= 1RNAP / 1-b-base,in this case, only one situation will

arise that will when promoter is started from 0, as both sides has same

speed so they will move equal distance hence collision will occur at mid

point. In this way 100% Transcriptional Interference is observed if 1RNAP/1-

b-base.

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Similarly, if P (L) = P(R)= 1RNAP/2-b-bases,in this, two cases will arise when

promoter is started from 0 and other from 1,both will move the equal

distance with same speed so here will be same 100% Transcriptional

Interference is observed after average of both case if 1RNAP/2-b-base.

Similarly, if P (L) = P(R) = 1RNAP/3-b-bases, in this, three cases will arise

when promoter is started from 0, then from 1, and in other from 2. Hence

after taking average of all three cases, approximately 66.66%Transcriptional

Interference is observed if 1RNAP/3-b-base.

Again, if P (L) = P(R) = 1RNAP/4-b-bases, Hence after taking average of all 4

cases, 50% Transcriptional Interference is observed.

Again, if P (L) = P(R) = 1RNAP/5-b-bases, Hence after taking average of all 5

cases, 40% Transcriptional Interference is observed.

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Again, If P (L) = P(R) = 1RNAP/6-b-bases, Hence after taking average of all 6

bases, 33.33% Transcriptional Interference is observed.

And if P (L) = P(R) = 1RNAP/7-b-bases, Hence after taking average of all 7

cases, 28.58% Transcriptional Interference is observed.

So, these are the seven different cases depending on different X-b-bases

when there is equal strength of promoters on both sides which we observed

and performed.

Now make the Table of First case for all 7 different cases on Microsoft Excel

when equal strength of promoters.

Then plot the corresponding graph between Rate of transcription on X-axis

and Transcriptional Interference (%) on Y-axis.

Table1. When equal strength of promoters (P (L) = P(R) = 1RNAP / X- b-

base).

Rate of Transcription (X-axis) Transcriptional Interference (%) (Y-axis)

1 100.00 2 100.00 3 66.66 4 50.00 5 40.00 6 33.33

7 28.58

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Graph1. Rate of transcription (X- axis) Vs Transcriptional Interference (%)

[Y-axis].

When equal strength of promoters (P (L) = P(R) = 1RNAP / X- b-base).

CASE2= When there is unequal Strength of promoters i.e. (1RNAP/

1- b-base) on LHS side and (1RNAP/ X- b-base) on RHS side.

In first case when P(L)=P(R)= 1RNAP / 1-b-base on both sides, in

this case, only one situation will arise that will when promoter is started from 0, as

both sides has same speed so they will move equal distance hence collision will

occur at mid point. In this way 100% Transcriptional Interference is

observed if 1RNAP/1-b-base on both sides.

0

20

40

60

80

100

120

0 2 4 6 8

Equal Strenght Of Promoter

Equal Strenght Of Promoter

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In 2nd case when P (L) =1RNAP/1-b-base on LHS and

P(R)=1RNAP/2-b-base on RHS , two cases will arise in which on LHS side

TI=50% in Both case and on RHS side TI=100% in both case, hence after taking

average on LHS side of both case we get TI=50% and on RHS side we get

average TI=100%.

Similarly in 3rd case when P (L) = 1RNAP/1-b-base on LHS and

P(R)=1RNAP/3-b-base on RHS , three cases will arise in which on

LHS side TI=50% in all three cases and on RHS side TI=100% in all

three cases, hence after taking average on LHS side of both case

we get TI=50% and on RHS side we get average TI=100%.

Similarly in 4th case average TI=50% on LHS and average TI= 75%

on RHS side.

And in the 5th case we found average TI=50% on LHS and average

TI=60% on RHS side.

So, these are the five different cases depending on different X-b-

bases when there is unequal strength of promoters on both sides

which we observed and performed.

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Now make the Table of second case for all 5 different cases on

Microsoft Excel when unequal strength of promoters on both

sides.

Then plot the corresponding graph between Rate of transcription on X-axis

and Transcriptional Interference (%) on Y-axis for both LHS and RHS on the

same graph.

Table2. When unequal strength of promoters P (L) = 1RNAP / 1- b-base on

LHS and P(R) = 1RNAP/X-b-base on RHS.

Rate of Transcription (X-axis) Transcriptional Interference (%) (Y-axis) (LHS)

Transcriptional Interference (%) (Y-axis) (RHS)

1 100.00 100.00 2 50.00 100.00 3 50.00 100.00 4 50.00 75.00 5 50.00 60.00

Graph1. Rate of transcription (X- axis) Vs Transcriptional Interference (%)

[Y- axis].

When unequal strength of promoters P (L) = 1RNAP / 1- b-base on LHS and

P(R) =1RNAP/X-b-base on RHS.

0

20

40

60

80

100

120

0 1 2 3 4 5 6

Unequal Strength of promoter(LHS)

Unequal Strength of promoter(RHS)

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So, these are the graph of TI based on strength of

promoters, plotted on Excel.

Now we will see how these different processes occur

randomly in actual and observe the dynamics of rate of

transcription and see the determined average time for the

transcription by Using Probability Distribution on MATLAB

and Rough both.

Dynamics of rate of transcription: -

A) Uniform Transcription: - In this case N times transcription is occurred at a uniform interval of long time‘t0’ so if N times transcription is

occurred in t time then for 1 time it will take (t0/N) times for transcription. B) Random Transcription: - In this case N times transcription is

occurred at random interval when RNA comes and bind and then they form the open complex. In this way bursting are found during this transcription. And transcription is occurred randomly at and distance and random time. This is the figure which showing the uniform and random transcription.

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Three cases we will observe from this: -

1) RNAP comes & binds- Transcription at a random time(Poisson

Distribution)

2) Pause(Wait) – (open complex formation)(Normal Distribution)

3) Movement(Transcription)- Uniform Speed

MATLAB FILE CODE (M-file code) which is used here to calculate the random time of these cases for the transcription are found in an array upto 500 seconds.

% random time generation from Poisson Distribution Lambda = 1 R1 = random ('Poisson',1:500,1,500) for n=1:499 Sum = [R1(n)+R1(n+1)] end % random time generation from Normal Distribution mu=0.2 sigma=0.0002 r2=normrnd(0.2,0.0002,1,500) for m=1:499; sum1=(r2(m)+r2(m+1)) end

Now, taking the average time of transcription of both cases A and B.

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So these three cases are observed from this: -

4) RNAP comes & binds-

5) Pause(Wait) – (open complex formation)

6) Movement(Transcription)

Conclusion: - After the observation of dynamics of rate of transcription on

Rough and Excel, we performed Random Stimulation based on Probability

Distribution using the MATLAB for the dynamics of rate of transcription. We made

the array of average time upto 500 seconds which actually required for the

transcription in gene regulation and we concluded that transcription is occurred

randomly.

Transcriptional Interference occurs in most genomes and has probably

persisted during evolution because of its potential use in regulating

gene expression. A variety of mechanisms have been identified and

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successfully studied, at least in simple systems. A major aim is to exploit

mathematical modeling to characterize TI, and a major issue to explore further is

the fate of an elongating RNAP when it meets a DNA-bound obstacle, either

moving or stationary.

final summer internship report new_

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