Lecture 9 - Transcription Regulation in Eukaryotes | Molecular Biology (DNA) by ILLL, DU - Biotechnology Engineering (BT) PDF Download

 Page 1


 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 1 
 
 
 
 
 
 
 
 
 
 
 
NME-Zoology 
Subject: Molecular Biology 
Lesson: Transcription Regulation in Eukaryotes 
Lesson Developer: Dr. Sudhir Verma 
College/Dept: Zoology, Deen Dayal Upadhyaya College 
University of Delhi 
 
 
 
 
 
 
 
 
 
 
  
Page 2


 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 1 
 
 
 
 
 
 
 
 
 
 
 
NME-Zoology 
Subject: Molecular Biology 
Lesson: Transcription Regulation in Eukaryotes 
Lesson Developer: Dr. Sudhir Verma 
College/Dept: Zoology, Deen Dayal Upadhyaya College 
University of Delhi 
 
 
 
 
 
 
 
 
 
 
  
 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 2 
Table of Contents 
Introduction 
 Activation of gene structure 
 Transcription initiation level 
 Processing and transport of transcript 
Activation of gene structure 
 Chromatin and its states 
 Mechanisms of altering the states of chromatin: 
? Post-translational modifications of histones 
? Chromatin Remodelling 
   Sliding 
   Transfer 
? DNA Methylation 
Transcription initiation level 
Transcriptional Activators 
? DNA Binding Domains 
Homeodomain Proteins (Helix-turn-helix) 
Helix-loop-helix Proteins 
Zinc containing Domains 
Leucine-zipper Motif 
? Activating Regions 
? How these transcriptional activators work 
Recruitment of transcriptional machinery 
Recruitment of nucleosome modifiers 
? Distantly placed eukaryotic activators: How do they 
work? 
Loops 
Insulators 
Locus Control Regions (LCR) 
 Signal Integration and Combinatorial Control 
  Synergy and co-operativity 
 Transcriptional Repressors 
  Competition with activator for binding site 
  Inhibition of activator 
  Direct repression of transcriptional machinery 
  Indirect repression by recruiting nucleosome modifiers 
 How are transcriptional regulators controlled: ‘Regulating the 
 Regulators’: Signal transduction mechanisms 
 Mode of action on regulators, once signal is received 
  By masking or unmasking the activating region of activator 
  By modifying cytoplasm-nucleoplasm shuttling 
 Additional levels of complexity in eukaryotes 
  Co-activators and co-repressors  
Beyond transcription initiation: Regulating transcription elongation step 
Processing and transport of transcript 
 At the level of capping 
 At the level of splicing 
 At the level of polyadenylation 
 At the level of mRNA transport 
RNA mediated gene regulation 
 
Page 3


 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 1 
 
 
 
 
 
 
 
 
 
 
 
NME-Zoology 
Subject: Molecular Biology 
Lesson: Transcription Regulation in Eukaryotes 
Lesson Developer: Dr. Sudhir Verma 
College/Dept: Zoology, Deen Dayal Upadhyaya College 
University of Delhi 
 
 
 
 
 
 
 
 
 
 
  
 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 2 
Table of Contents 
Introduction 
 Activation of gene structure 
 Transcription initiation level 
 Processing and transport of transcript 
Activation of gene structure 
 Chromatin and its states 
 Mechanisms of altering the states of chromatin: 
? Post-translational modifications of histones 
? Chromatin Remodelling 
   Sliding 
   Transfer 
? DNA Methylation 
Transcription initiation level 
Transcriptional Activators 
? DNA Binding Domains 
Homeodomain Proteins (Helix-turn-helix) 
Helix-loop-helix Proteins 
Zinc containing Domains 
Leucine-zipper Motif 
? Activating Regions 
? How these transcriptional activators work 
Recruitment of transcriptional machinery 
Recruitment of nucleosome modifiers 
? Distantly placed eukaryotic activators: How do they 
work? 
Loops 
Insulators 
Locus Control Regions (LCR) 
 Signal Integration and Combinatorial Control 
  Synergy and co-operativity 
 Transcriptional Repressors 
  Competition with activator for binding site 
  Inhibition of activator 
  Direct repression of transcriptional machinery 
  Indirect repression by recruiting nucleosome modifiers 
 How are transcriptional regulators controlled: ‘Regulating the 
 Regulators’: Signal transduction mechanisms 
 Mode of action on regulators, once signal is received 
  By masking or unmasking the activating region of activator 
  By modifying cytoplasm-nucleoplasm shuttling 
 Additional levels of complexity in eukaryotes 
  Co-activators and co-repressors  
Beyond transcription initiation: Regulating transcription elongation step 
Processing and transport of transcript 
 At the level of capping 
 At the level of splicing 
 At the level of polyadenylation 
 At the level of mRNA transport 
RNA mediated gene regulation 
 
 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 3 
Introduction 
? The various kinds of cells in multi-cellular higher eukaryotes are distinguished 
by phenotypic differences. 
? These differences are governed by differences in gene expression.  
? In other words, each cell expresses only a selective subset of genes as per its 
requirement.  
? Gene expression is regulated by certain rules and mechanisms which operate 
at a number of steps during expression of genetic material. Some of the 
potential regulatory check points are shown below: 
 
Figure 01: Different control points during sequential steps of gene 
expression. 
Source: Author 
Within the scope of current topic, we will focus on the control mechanisms for 
regulation of transcription only. The post-transcriptional controls may include 
translation and other RNA mediated regulation of gene expression, which is beyond 
the scope of current chapter. Let us briefly understand what all it includes: 
Activation of gene structure:  
? Before the actual transcription starts, the pre-requisite is that a particular 
gene is „transcribable?. It can be understood as: before appearing in an 
entrance exam, we need to check whether we are eligible to appear in that 
exam or not. That „eligible? nature is called as „active? state of a gene.  
? A particular gene can exist in active or inactive state. The accessibility of the 
gene to the transcriptional machinery defines whether a gene is active or not. 
As we know, in order to accommodate around 2 meters long DNA of 
mammalian diploid cell in the tiny nucleus of a few micron dimensions, there 
exist higher orders of DNA packaging. On one hand where this condensation 
is pre-requisite for accommodation, there are problems associated with other 
Page 4


 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 1 
 
 
 
 
 
 
 
 
 
 
 
NME-Zoology 
Subject: Molecular Biology 
Lesson: Transcription Regulation in Eukaryotes 
Lesson Developer: Dr. Sudhir Verma 
College/Dept: Zoology, Deen Dayal Upadhyaya College 
University of Delhi 
 
 
 
 
 
 
 
 
 
 
  
 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 2 
Table of Contents 
Introduction 
 Activation of gene structure 
 Transcription initiation level 
 Processing and transport of transcript 
Activation of gene structure 
 Chromatin and its states 
 Mechanisms of altering the states of chromatin: 
? Post-translational modifications of histones 
? Chromatin Remodelling 
   Sliding 
   Transfer 
? DNA Methylation 
Transcription initiation level 
Transcriptional Activators 
? DNA Binding Domains 
Homeodomain Proteins (Helix-turn-helix) 
Helix-loop-helix Proteins 
Zinc containing Domains 
Leucine-zipper Motif 
? Activating Regions 
? How these transcriptional activators work 
Recruitment of transcriptional machinery 
Recruitment of nucleosome modifiers 
? Distantly placed eukaryotic activators: How do they 
work? 
Loops 
Insulators 
Locus Control Regions (LCR) 
 Signal Integration and Combinatorial Control 
  Synergy and co-operativity 
 Transcriptional Repressors 
  Competition with activator for binding site 
  Inhibition of activator 
  Direct repression of transcriptional machinery 
  Indirect repression by recruiting nucleosome modifiers 
 How are transcriptional regulators controlled: ‘Regulating the 
 Regulators’: Signal transduction mechanisms 
 Mode of action on regulators, once signal is received 
  By masking or unmasking the activating region of activator 
  By modifying cytoplasm-nucleoplasm shuttling 
 Additional levels of complexity in eukaryotes 
  Co-activators and co-repressors  
Beyond transcription initiation: Regulating transcription elongation step 
Processing and transport of transcript 
 At the level of capping 
 At the level of splicing 
 At the level of polyadenylation 
 At the level of mRNA transport 
RNA mediated gene regulation 
 
 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 3 
Introduction 
? The various kinds of cells in multi-cellular higher eukaryotes are distinguished 
by phenotypic differences. 
? These differences are governed by differences in gene expression.  
? In other words, each cell expresses only a selective subset of genes as per its 
requirement.  
? Gene expression is regulated by certain rules and mechanisms which operate 
at a number of steps during expression of genetic material. Some of the 
potential regulatory check points are shown below: 
 
Figure 01: Different control points during sequential steps of gene 
expression. 
Source: Author 
Within the scope of current topic, we will focus on the control mechanisms for 
regulation of transcription only. The post-transcriptional controls may include 
translation and other RNA mediated regulation of gene expression, which is beyond 
the scope of current chapter. Let us briefly understand what all it includes: 
Activation of gene structure:  
? Before the actual transcription starts, the pre-requisite is that a particular 
gene is „transcribable?. It can be understood as: before appearing in an 
entrance exam, we need to check whether we are eligible to appear in that 
exam or not. That „eligible? nature is called as „active? state of a gene.  
? A particular gene can exist in active or inactive state. The accessibility of the 
gene to the transcriptional machinery defines whether a gene is active or not. 
As we know, in order to accommodate around 2 meters long DNA of 
mammalian diploid cell in the tiny nucleus of a few micron dimensions, there 
exist higher orders of DNA packaging. On one hand where this condensation 
is pre-requisite for accommodation, there are problems associated with other 
 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 4 
vital cellular processes. Transcription, translation, replication and repair 
mechanisms employ enzymatic machinery which needs accessibility to DNA. 
Thus, these conflicting states of condensation-decondensation define active-
inactive state of a gene, respectively. 
Transcription initiation level:  
? Transcription initiation refers to binding of transcriptional machinery to 
promoter of that particular gene.  
? Transcriptional machinery in eukaryotes comprises of RNA polymerase 
enzyme, mediator complex, other general transcription factors and accessory 
proteins, if any.  
? Various regulatory proteins (activators, repressors, insulators, nucleosome 
modifiers etc.) control this initiation process.  
Processing and transport of transcript:  
? Polyadenylation at 3?end and capping at 5? end of primary transcript, splicing 
(excision of introns) and export of transcript from nucleus to cytosol for 
further gene expression (i.e. translation) are some of the processes that are 
controlled at post-initiation level in transcriptional regulation.  
Out of all these possible levels mentioned above, majority of regulatory events takes 
place at transcription initiation level and it makes sense for the cell to stop the gene 
expression by blocking the very first step i.e. transcription. Why a cell would waste 
its energy in making an mRNA for the protein that is not required.  
 
ACTIVATION OF GENE STRUCTURE 
Chromatin and its states: 
Chromatin is defined as nucleo-protein complex, comprised of DNA and chromosomal 
proteins (histones and non-histones).The physiological purpose of this association is 
to confine the large amount of DNA within the nuclear limits. This level of compaction 
is achieved by dynamic levels of DNA-protein organization. DNA in its associated 
form with protein is inaccessible to enzymatic machinery for carrying out vital 
cellular functions like replication, repair, transcription and translation. This repressive 
effect of chromatin on cellular events is temporally and spatially altered by dynamic 
nature of chromatin. Chromatin can unwind with the help of enzymes and protein 
complexes within the cell.  
Thus depending on cellular need, two alternate states of chromatin exist: condensed 
heterochromatin and open euchromatin state. The heterochromatinized state is 
transcriptionally inactive and euchromatinized state is transcriptionally active. 
Mechanisms of altering the states of chromatin: 
? Post-translational modifications of histones 
The fundamental structural unit of chromatin is called nucleosome. It comprises of 
~200bp of DNA wrapped over a core histone octamer. 146-147bp of DNA i.e. core 
Page 5


 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 1 
 
 
 
 
 
 
 
 
 
 
 
NME-Zoology 
Subject: Molecular Biology 
Lesson: Transcription Regulation in Eukaryotes 
Lesson Developer: Dr. Sudhir Verma 
College/Dept: Zoology, Deen Dayal Upadhyaya College 
University of Delhi 
 
 
 
 
 
 
 
 
 
 
  
 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 2 
Table of Contents 
Introduction 
 Activation of gene structure 
 Transcription initiation level 
 Processing and transport of transcript 
Activation of gene structure 
 Chromatin and its states 
 Mechanisms of altering the states of chromatin: 
? Post-translational modifications of histones 
? Chromatin Remodelling 
   Sliding 
   Transfer 
? DNA Methylation 
Transcription initiation level 
Transcriptional Activators 
? DNA Binding Domains 
Homeodomain Proteins (Helix-turn-helix) 
Helix-loop-helix Proteins 
Zinc containing Domains 
Leucine-zipper Motif 
? Activating Regions 
? How these transcriptional activators work 
Recruitment of transcriptional machinery 
Recruitment of nucleosome modifiers 
? Distantly placed eukaryotic activators: How do they 
work? 
Loops 
Insulators 
Locus Control Regions (LCR) 
 Signal Integration and Combinatorial Control 
  Synergy and co-operativity 
 Transcriptional Repressors 
  Competition with activator for binding site 
  Inhibition of activator 
  Direct repression of transcriptional machinery 
  Indirect repression by recruiting nucleosome modifiers 
 How are transcriptional regulators controlled: ‘Regulating the 
 Regulators’: Signal transduction mechanisms 
 Mode of action on regulators, once signal is received 
  By masking or unmasking the activating region of activator 
  By modifying cytoplasm-nucleoplasm shuttling 
 Additional levels of complexity in eukaryotes 
  Co-activators and co-repressors  
Beyond transcription initiation: Regulating transcription elongation step 
Processing and transport of transcript 
 At the level of capping 
 At the level of splicing 
 At the level of polyadenylation 
 At the level of mRNA transport 
RNA mediated gene regulation 
 
 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 3 
Introduction 
? The various kinds of cells in multi-cellular higher eukaryotes are distinguished 
by phenotypic differences. 
? These differences are governed by differences in gene expression.  
? In other words, each cell expresses only a selective subset of genes as per its 
requirement.  
? Gene expression is regulated by certain rules and mechanisms which operate 
at a number of steps during expression of genetic material. Some of the 
potential regulatory check points are shown below: 
 
Figure 01: Different control points during sequential steps of gene 
expression. 
Source: Author 
Within the scope of current topic, we will focus on the control mechanisms for 
regulation of transcription only. The post-transcriptional controls may include 
translation and other RNA mediated regulation of gene expression, which is beyond 
the scope of current chapter. Let us briefly understand what all it includes: 
Activation of gene structure:  
? Before the actual transcription starts, the pre-requisite is that a particular 
gene is „transcribable?. It can be understood as: before appearing in an 
entrance exam, we need to check whether we are eligible to appear in that 
exam or not. That „eligible? nature is called as „active? state of a gene.  
? A particular gene can exist in active or inactive state. The accessibility of the 
gene to the transcriptional machinery defines whether a gene is active or not. 
As we know, in order to accommodate around 2 meters long DNA of 
mammalian diploid cell in the tiny nucleus of a few micron dimensions, there 
exist higher orders of DNA packaging. On one hand where this condensation 
is pre-requisite for accommodation, there are problems associated with other 
 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 4 
vital cellular processes. Transcription, translation, replication and repair 
mechanisms employ enzymatic machinery which needs accessibility to DNA. 
Thus, these conflicting states of condensation-decondensation define active-
inactive state of a gene, respectively. 
Transcription initiation level:  
? Transcription initiation refers to binding of transcriptional machinery to 
promoter of that particular gene.  
? Transcriptional machinery in eukaryotes comprises of RNA polymerase 
enzyme, mediator complex, other general transcription factors and accessory 
proteins, if any.  
? Various regulatory proteins (activators, repressors, insulators, nucleosome 
modifiers etc.) control this initiation process.  
Processing and transport of transcript:  
? Polyadenylation at 3?end and capping at 5? end of primary transcript, splicing 
(excision of introns) and export of transcript from nucleus to cytosol for 
further gene expression (i.e. translation) are some of the processes that are 
controlled at post-initiation level in transcriptional regulation.  
Out of all these possible levels mentioned above, majority of regulatory events takes 
place at transcription initiation level and it makes sense for the cell to stop the gene 
expression by blocking the very first step i.e. transcription. Why a cell would waste 
its energy in making an mRNA for the protein that is not required.  
 
ACTIVATION OF GENE STRUCTURE 
Chromatin and its states: 
Chromatin is defined as nucleo-protein complex, comprised of DNA and chromosomal 
proteins (histones and non-histones).The physiological purpose of this association is 
to confine the large amount of DNA within the nuclear limits. This level of compaction 
is achieved by dynamic levels of DNA-protein organization. DNA in its associated 
form with protein is inaccessible to enzymatic machinery for carrying out vital 
cellular functions like replication, repair, transcription and translation. This repressive 
effect of chromatin on cellular events is temporally and spatially altered by dynamic 
nature of chromatin. Chromatin can unwind with the help of enzymes and protein 
complexes within the cell.  
Thus depending on cellular need, two alternate states of chromatin exist: condensed 
heterochromatin and open euchromatin state. The heterochromatinized state is 
transcriptionally inactive and euchromatinized state is transcriptionally active. 
Mechanisms of altering the states of chromatin: 
? Post-translational modifications of histones 
The fundamental structural unit of chromatin is called nucleosome. It comprises of 
~200bp of DNA wrapped over a core histone octamer. 146-147bp of DNA i.e. core 
 Transcription Regulation in Eukaryotes 
 Institute of Lifelong Learning, University of Delhi 5 
DNA along with eight histone proteins forms the nucleosome core particle and 
remaining 53-54bp of DNA (linker DNA) connects the adjacent core particles. Histone 
H1 stabilizes the nucleosome structure. Histones are small molecular weight (11-
22kDa) proteins that constitute major protein component of eukaryotic chromatin. 
These proteins are rich in lysine and arginine amino acids, and hence are basic in 
nature with positive charge at physiological pH.  
Five major types of histones are present in all somatic cells of eukaryotes: H1 (the 
linker histone) and H2A, H2B, H3, H4 (the core histones). The classification of 
histones is based on their lysine to arginine content ratio.  
Histone Histone 
type 
% of lysine 
and arginine 
Units per 
nucleosome 
H1 Linker 32 01 
H2A Core 20 02 
H2B Core 22 02 
H3 Core 23 02 
H4 Core 24 02 
 
   Table 01: Histone types and their properties 
Source: Modified from: Molecular Biology of the Gene by Watson et. al. 6
th
 
edition (2008), Cold Spring Harbor Laboratory Press 
? Structurally, all the core histones contain three different regions: a conserved 
histone fold (globular) domain, a-helical histone fold extension unique to the 
different histones, and the unstructured, labile N- and C- terminal tail.  
? The tails (at N-termini of all core histones and at C-terminal of histone H2A) 
protrude out of nucleosome core. Histone tails contain sites for covalent, 
post-translational modifications and thus, provide a binding/ docking platform 
to diverse range of protein factors.  
? These modifications and/ or bound factors modulate the DNA accessibility to 
enzymatic machinery by altering the chromatin. The alteration of chromatin 
state by various enzyme complexes without expenditure of ATP is termed as 
„ATP-independent chromatin remodelling?. These mechanisms include covalent 
post-translational modifications of histone tails primarily, and at times 
globular domains as well. A number of such modifications are known such as, 
acetylation, methylation, phosphorylation, ADP-ribosylation, ubiquitinylation, 
sumoylation, propionylation, butyrylation, proline isomerization, glycosylation, 
citrullination, biotinylation etc.  
? These modifications are histone site-specific in nature but, same amino acid 
site can have more than one modification e.g. Histone H3 lysine 9 can be both 
acetylated and methylated.  
? Different enzymes are available which write these modifications that are read 
by various reader molecules followed by removal by so called eraser 
molecules.   
? DNA-histone interaction is altered by the charge provided by these 
modifications that alters the packaging of chromatin and in turn, expression