1. Genes-Characteristics and Function

Genes are the basic unit of inheritance containing the information for physical and biological traits. They contain the information necessary for living cells to survive and reproduce . Genes are regions of the DNA .They are passed from parents to offspring. They code for specific proteins or segments of proteins. Humans have approximately 20,000 protein coding genes . These  20,000 protein coding genes are encoded only 1.5% of the entire human genome. Not all genes code only for proteins , some genes encode information for making an RNA molecule that functions other than directly coding for a protein.

Genes in genome of eukaryotes consist of exons and introns. [pb_glossary id="878"]Exons[/pb_glossary] are the protein coding regions of mRNA. These are the regions that contain information for making a protein whereas other regions of the RNA are non-coding and these regions are called [pb_glossary id="879"]introns[/pb_glossary]

The majority of human genes have two or more possible [pb_glossary id="880"]alleles[/pb_glossary], which are alternative forms of a gene. Differences in alleles account for the considerable genetic variation among people. In fact, most human genetic variation is the result of differences in individual DNA bases within alleles.

"Gene_Intron_Exon" by Smedlib via Wikimedia commons is licensed under CC BY-SA 4.0

Genomes can be twisted in the same direction as the double helix or positively super coiled . Alternatively genome can be twisted in the opposite direction of the double helix or       negatively supercoiled . Most bacterial genomes are negatively supercoiled . This is achieved with the help of a protein called HU,  which is the most abundant protein in the nucleoid. The protein Hu along with an enzyme called topoisomerase I to bind DNA and introduce sharp bends in the chromosome. This  generates  the tension necessary for negative supercoiling.

Another protein called  Integration Host Factor (IHF), can bind to specific sequences within the genome and introduce additional bends. The folded DNA is then organized into a variety of conformations that are supercoiled and wound around tetramers of the HU protein. DNA topoisomerase I, DNA gyrase, and other proteins help maintain the supercoils.

The DNA molecules of [pb_glossary id="899"]Archaea, [/pb_glossary]a taxonomic domain composed of single -celled ,non bacterial prokaryotes that share many similarities with eukaryotes. Their DNA can be negatively supercoiled, positively supercoiled, or not supercoiled. Archaeans are the only group of prokaryotes that use eukaryote-like histones, rather than the architectural proteins described above, to condense their DNA molecules. It is believed that the acquisition of histones by archaeans had paved the way for the evolution of larger and more complex eukaryotic cells.

"Prokaryote cell" by Mariana Ruiz Villarreal, LadyofHatsDifference via Wikimedia commons is licensed under CC BY-SA 3.0 

Humans have 23 pairs of chromosomes , of which 22 pairs are [pb_glossary id="901"]autosomes[/pb_glossary] or body chromosomes  . Autosomes  look the same in both males and females . Apart from autosomes there is a  pair of  [pb_glossary id="902"]allosome [/pb_glossary]or sex chromosomes which differ between males and females.

Each pair contains two chromosomes, one acquired from each parent, which means that children inherit half of their chromosomes from their mother and half from their father. Chromosomes can be seen through a microscope when the nucleus dissolves during cell division.

The matched pairs of chromosomes in a diploid organism are called homologous chromosomes.[pb_glossary id="904"] Homologous chromosomes[/pb_glossary] are of  the same length and have genes in exactly the same location, or locus.A somatic cell contains two matched sets of chromosomes, a configuration known as diploid ( 2n)  where ' n' represent a single set of chromosomes;

Gametes, or sex cells  (eggs and sperm )  contain only one set of 23 chromosomes (n)  and are designated  as haploid.

The variation of individuals within a species is caused by the specific combination of the genes inherited from both parents. Genes are expressed as characteristics of the organism . Each characteristic may have different variants called traits . Traits are caused by differences in the DNA sequence for a gene. An organism’s traits are determined largely by the genes     inherited from each parent and also by the environment that they experience.

"Chromosomes" by Andreas Bolzer, Gregor Kreth, Irina Solovei, Daniela Koehler, Kaan Saracoglu, Christine Fauth, Stefan Müller, Roland Eils, Christoph Cremer, Michael R. Speicher, Thomas Cremer, is licensed under CC BY-SA 2.5

Types of Chromosomes 

• Telocentric– Telocentric chromosomes have the centromere at the end of the chromosome . Here centromere occupy the terminal position. Thus, the chromosome in this case  has just one arm. Such rod shaped chromosomes are found in species such as mice. 
• Acrocentric- In this type ,centromere occupying a sub-terminal position  making one arm very long and the other very short. Human chromosomes 13, 15, 21, and 22 are acrocentric. 
• Sub-metacentric– This type of chromosomes are with centromere slightly away from the mid-point. The two arms are unequal. Human chromosomes 4 through 12 are sub-metacentric. 
• Metacentric- Metacentric chromosomes are V-shaped . The  centromere lies in the middle of chro­mosome or in the center.  The two arms are almost equal. Human chromosome 1 and 3 are metacentric.

• Monocentric- Chromosomes are with one centromere. 
• Dicentric-Chromosomes  with two centromeres. 
• Polycentric- Chromosomes with more than two centromeres. 
• Acentric- Such chromosomes are without centromere. These are freshly broken segments of chromosomes. 
• Diffused or non-located- These are chromosomes with indistinct centromere diffused throughout the length of chromosome.

Functions of Chromosomes 

• Chromosomes are the essential unit for cellular division
• Chromosomes must be replicated, divided, and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny.
• Chromosomes protect the DNA from damage
• The dynamics of  chromosome structural changes regulate gene expression.
• Chromosomal recombination plays a vital role in genetic diversity.

[pb_glossary id="909"]Histones [/pb_glossary]are basic proteins. Histones are enriched with basic lysine and arginine residues, Their positive charges enables  them to associate with the negatively charged DNA ( due to the phosphates , PO4 ^- ) . The histones function as spools for the thread-like DNA to wrap around. Histones may be chemically modified through the action of enzymes to regulate gene expression. Core histones share a structural motif known as the histone fold domain, formed by three α-helices connected by two loops.

Types of Histones 

Basically histones are of two category namely

Core histone  which includes  H2A, H2B, H3 & H4 and  Linker histone  which includes H1.

H2A is the core histone with the largest number of variants. The histone H2A variants which are found in most eukaryotes are H2AZ and H2AX. Phosphorylation of H2AX has been found to aid in the recruitment of proteins involved in DNA repair

Histone H2B variants are few in number and  have specialized roles in chromatin compaction during gametogenesis

Histone H3 variants include H3.3, CenHS and H3.4. H3.3  is found in transcriptionally active chromatin 

Histone H4 is the most highly conserved histone. H4 makes extensive contacts with the other three core histones in the nucleosome core particle 

Gene Organization in Eukaryotes 

The large eukaryotic genome or DNA approximately 2 m in length is packed  in to a nucleus of roughly 10 µm diameter through an orderly packaging.

Nucleosomes

Nucleosomes are the basic unit of DNA packaging in eukaryotes and represent the first level of chromatin organization. A nucleosome consist of a segment of DNA wound around a   protein core called -histone octamer. This structure is often compared to thread wrapped around a spool.

The nucleosome core particle consists of approximately 146- 147 base pairs of DNA  wrapped  around a histone octamer , consisting of 2 copies each of the core histones namely  H2A, H2B, H3, and H4.The octamer structure is a four-helix bundle comprised of two H3-H4 and two H2A-H2B dimers.

Nucleosome cores are separated by linker DNA of variable length ( 20 to 80 base pairs of linker DNA ) and are associated with the linker histone H1. Nucleosomes connected by a DNA linker of variable  forms a 10-nm beads-on-a-string array. 

Nucleosomes  pack DNA in a sequence-independent fashion. The core histones make contact with the DNA primarily through three to six hydrogen bonds between the protein main chain amides and the DNA phosphate backbone.

 Nucleosome particles can be modified in their composition, structure and location by chromatin remodeling complexes and regulates  gene expression. Nucleosomes are folded through a series of successively higher order structures to eventually form a chromosome. A nucleosome plus histone H1 is referred to as[pb_glossary id="912"] Chromatosome[/pb_glossary] 

The 30 nm Fibre 

The chromatosome fold  up to a 30 nm fibre .Nucleosome core particles can stack on top of each other to form extended structures. These stacks can adopt a gentle helical configuration.  The 30 nm fibre  consists of a helical array of nucleosomes, each comprising a core particle wrapping ∼146 or 147 base pairs (bp) of DNA associated with a linker histone. Secondary structures to chromatin are demonstrated by the solenoid model and the zigzag model. The solenoid model consists of tightly wound nucleosomes containing 6 nucleosomes per turn in a regular, spiral configuration . The zigzag model is a bit looser form of chromatin with irregular configuration. In this model, nucleosomes have little face-to-face contact.

Nuclear Scaffold 

Higher order packaging consisting of loops of the 30-nm fibers attached to a proteinaceous nuclear scaffold by an interaction between the scaffold and specific DNA sequences called [pb_glossary id="914"]Scaffold/matrix attachment regions (S/MARs[/pb_glossary]) . These are DNA elements that serve to compartmentalize the chromatin into structural and functional domains.

Loops are formed averaging about 300nm in length.  The 300nm fibre are compressed and folded  producing a 250 nm wide fibre .

Upon tight coiling of the 250nm wide fiber the chromatid of chromosome is formed. 

"Organization of Eukaryotic chromosomes " by CNX Open Stax Via Wikimedia commons is licensed under CC BY 4.0

""How DNA is Packaged ?"" by DNA Learning Center is licensed under CC BY 3.0

Gene Expression

The process of turning on a gene to produce RNA and protein is called [pb_glossary id="917"]gene expression.[/pb_glossary] For a cell to function properly appropriate  proteins must be synthesized at the appropriate  time. Whether in a simple unicellular organism  or a complex multi-cellular organism, each cell controls  when and how its genes are expressed.

Central Dogma of Molecular Biology -Information flow in biological systems

The central dogma of molecular biology  explains the flow of genetic information within a biological system. It is often stated as "DNA makes RNA, and RNA makes protein" .

•  DNA can be copied to DNA through a molecular process called DNA Replication. Instructions for making  proteins with the correct sequence of amino acids are encoded in DNA.
•  This information in DNA can be copied into RNA  through the molecular process called  [pb_glossary id="919"]Transcription [/pb_glossary]by which the information in the DNA of every cell is converted into small, portable RNA messages.
•  Proteins can be synthesized using the information in mRNA as a template through the molecular process called [pb_glossary id="921"]Translation.[/pb_glossary] During translation, the RNA messages travel from DNA  in the nucleus to the ribosomes in the cytoplasm where they are ‘read’ to make specific proteins.

Prokaryotic Gene regulation

 Regulation of Gene expression  in Prokaryotes

Operons

• Structural genes - genes that encode proteins used in metabolism or biosynthesis or that play a structural role in the cell and 
• Regulatory elements-  includes the promoter and the region surrounding the promoter and the genes that code for the regulatory proteins

•  Repressors- are proteins that suppress transcription of a gene in response to an external stimulus,
• Activators - are proteins that increase the transcription of a gene in response to an external stimulus and
• Inducers - are small molecules that either activate or repress transcription depending on the needs of the cell and the availability of substrate.

Prokaryotic RNA Polymerase

• The process of transcription begins when an enzyme called RNA Polymerase  which attaches to the template DNA strand and begins to catalyze production of complementary RNA. Prokaryotes use the same RNA polymerase to transcribe all of their genes.
• In E. coli, the polymerase is composed of five polypeptide subunits, comprising the Core enzyme  and the holoenzyme .
• The five subunits include
  •  two α-subunits  :  are necessary to assemble the polymerase on the DNA
  •  β-subunit :  binds to the ribonucleoside triphosphate that will become part of the nascent “recently born” mRNA molecule;
  •  β′ - subunit  : binds the DNA template strand.
  •  subunit  σ  : is involved only in transcription initiation. It confers transcriptional specificity such that the polymerase begins to synthesize mRNA from an appropriate initiation site. Without σ, the core enzyme would transcribe from random sites

The  α, α, β, and β′ comprise the polymerase core enzyme. The polymerase comprised of all five subunits is called the [pb_glossary id="929"]holoenzyme[/pb_glossary] (a holoenzyme is a biochemically active and is comprised of an enzyme and its coenzyme).

The Process of Prokaryotic Transcription

Template or Non coding strand 

DNA is double-stranded, but only one strand serves as a template for transcription at any given time. This[pb_glossary id="925"] template strand[/pb_glossary] is called the noncoding strand.

Non template or Coding strand 

The other strand of DNA which is not serving as template is called the  [pb_glossary id="927"]nontemplate[/pb_glossary]  or coding strand . This strand is referred to as the coding strand because its sequence will be the same as that of the new RNA molecule. In most organisms, the strand of DNA that serves as the template for one gene may be the nontemplate strand for other genes within the same chromosome.

Upstream and Down stream Sequences 

These are  conventions used to describe features of a DNA sequence, gene or mRNA related to the position and direction (5' to 3') of transcription by RNA polymerase or translation by the ribosome.

Downstream (or 3' to) is in the direction of transcription (or translation) whereas

Upstream (5' to) is in the direction from which the polymerase (or ribosome) has come.( Upstream DNA sequences are before the start site of transcription)

Image "Transcription Bubble" by Ristinn via Wikimedia Commons is licensed under CC BY-SA 4.0

INITIATION

The first step in transcription is initiation, when the RNA pol binds to the DNA upstream (5′) of the gene at a specialized sequence called a promoter .

Prokaryotic Promoters

• The DNA sequence onto which the proteins and enzymes involved in transcription bind to initiate the process is called a[pb_glossary id="932"] promoter[/pb_glossary]. Promoters usually exist upstream of the genes they regulate. The specific sequence of a promoter determines whether the corresponding gene is transcribed all of the time, some of the time, or hardly at all. The structure and function of a prokaryotic promoter is relatively simple 
• The -10 consensus sequence or the Pribnow box  : In prokaryotes, most genes have a sequence called the Pribnow box, with the consensus sequence TATAAT positioned about ten base pairs away from the site . This serves as the location of transcription initiation. Not all Pribnow boxes have this exact nucleotide sequence; these nucleotides are simply the most common ones found at each site.
• The -35 consensus sequence : Many genes also have the consensus sequence TTGCCA at -35  position, upstream of the start site
• Upstream element  : an A-T rich region 40 to 60 nucleotides upstream that enhances the rate of transcription

"Prokaryotic Promoter" Image from Biology 2e (2nd edition), an OpenStax resource licensed under CC BY 4.0

 The process of transcription starts with the binding of  the RNA pol "holoenzyme" binding to the template DNA and  unwinds  the DNA double helix in order to facilitate access to the gene. The sigma subunit conveys promoter specificity to RNA polymerase; that is, it instructs the  RNA polymerase where to bind. There are a number of different sigma subunits that bind to different promoters. These sigma subunits assist in turning genes on and off as conditions change.

Elongation

Once transcription is initiated, the DNA double helix unwinds and RNA polymerase reads the template strand. Elongation phase begins with the release of the σ subunit from the polymerase. The dissociation of σ allows the core enzyme to proceed along the DNA template, synthesizing mRNA in the 5′ to 3′ direction .

Nucleotides  are added to the 3′ end of the growing chain . At a temperature of 37⁰ C new nucleotides are added at an estimated rate of about 42-54 nucleotides per second in bacteria. As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it. The base pairing between DNA and RNA is not stable enough to maintain the stability of the mRNA synthesis components. Instead, the RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongation is not interrupted prematurely.

"Process of transcription " by NHS National Genetics and Genomics Education Centre. is licensed under CC BY 2.0 

Termination

Once a gene is transcribed, the prokaryotic polymerase needs to be instructed to dissociate from the DNA template and liberate the newly made mRNA. Depending on the gene being transcribed, there are two kinds of termination signals namely :

1. Rho-dependent or Extrinsic termination : Termination is controlled by the rho protein.The rho protein tracks along behind the polymerase on the growing mRNA chain. Near the end of the gene, the polymerase encounters a run of G nucleotides on the DNA template and it stalls. As a result, the rho protein collides with the polymerase. Rho unwinds the DNA-RNA hybrid formed during transcription and releases the mRNA from the transcription bubble.
2. Rho-independent or Intrinsic termination is controlled by  C–G nucleotide sequences in the DNA template strand at the end of the gene being transcribed. Due to the self complementary nature of the C-G nucleotides . The mRNA folds back on itself, and form a stable hairpin . This causes the polymerase to stall and  it begins to transcribe a region rich in A–T nucleotides. The complementary U–A region of the mRNA transcript forms only a weak interaction with the template DNA. This fact together  with the stalled polymerase, induces enough instability for the core enzyme to break away and liberate the new mRNA transcript.

Upon termination, the process of transcription is complete. By the transcription would end the prokaryotic transcript would  have already commenced the   synthesis of numerous copies of the encoded protein .

"Prokaryotic Terminators " by Oalnafo via Wikimedia Commons is licensed under CC BY-SA 3.0

Eukaryotic transcription is carried out in the nucleus of the cell . Eukaryotes also employ three different polymerases that each transcribe the various subset of genes. The process take place in three stages as 

1. Initiation
2. Elongation
3. Termination.

 Eukaryotes require proteins, called transcription factors, to first bind to the promoter region which then help to recruit the appropriate polymerase.

Eukaryotic RNA Polymerases 

The  three different RNA polymerases in eukaryotes, each  comprise  of 10 subunits or more and require a distinct set of transcription factors to bring the RNA polymerase to the DNA template.

RNA polymerase I is located in the nucleolus. Transcribe the genes for Ribosomal RNA ( r RNA)- transcribes the 28S, 18S, and 5.8S rRNA genes. . These are structural RNAs has they have a cellular role but are translated in to protein.. The rRNAs are components of the ribosome and are essential to the process of translation. RNA polymerase I synthesizes most of the rRNAs.

RNA polymerase II is located in the nucleus.  Synthesizes all  pre-mRNAs and small nucleolar RNAs (snoRNAs) involved in rRNA processing and small nuclear RNAs (snRNAs) involved in mRNA processing, except for U6 snRNA. The  eukaryotic pre-mRNAs undergo extensive processing after transcription but before translation.

RNA polymerase III is also located in the nucleus. This polymerase transcribes a variety of structural RNAs including tRNA, 5S rRNA, U6 snRNA, and the 7S RNA associated with the signal recognition particle (SRP). The tRNAs have a critical role serve as the adaptor molecules between the mRNA template and the growing polypeptide chain. Small nuclear RNAs have a variety of functions, including “splicing” pre-mRNAs and regulating transcription factors.

Each of the types of RNA polymerase recognizes a different promoter sequence and requires different transcription factors.

The RNA polymerase transcribes one strand, the antisense (-) strand, of the DNA template. RNA synthesis does not require a primer and occurs in the 5’ → 3’ direction .

The RNA polymerase catalyzes a nucleophilic attack by the 3'-OH of the growing RNA chain on the alpha-phosphorus atom of an incoming ribonucleoside 5'-triphosphate.

All three of the nuclear RNA polymerases are complex enzyme . Each consisting of 8 to 14 different subunit . Although they recognize different promoters and transcribe distinct classes of genes, they share several common features.

The two largest subunits of all three eukaryotic RNA polymerases are related to the β and β′subunits of the single E. coli RNA polymerase.

In addition, all three eukaryotic polymerases have common five subunits .

• Elongation includes repetitive cycles of decoding the m RNA , peptide bond formation, and translocation.
• Elongation begins as soon as the second codon of the ORF becomes accessible for reading by the next amino acylated tRNAs 
•  Elongation is facilitated by  translation factors EF-Tu, EF-G ,EF-Ts EF-P and EF-4.

• The second  codon  in the P site is recognized by aminoacylated -tRNAs.  A ternary complex of  aa-tRANA ,EF-Tu and GTP is attached to the P site .This interaction triggers GTP hydrolysis by EF-Tu. After Pi release, EF-Tu rearranges into the guanosine diphosphate (GDP)-bound form and releases the aa-tRNA.
• The amino group of the amino acid attached to the A-site tRNA  makes a peptide bond with the carboxyl group of the amino acid attached to the P-site tRNA. This reaction is catalyzed by peptidyl transferase, an RNA-based ribozyme that is integrated into the 50S ribosomal subunit. Now the A site carries a peptide comprising of two amino acids and leaves the uncharged t RNA in the p site .
• During elongation the ribosomes move one codon along the mRNA in a process called Translocation. Translocation is promoted by EF-G at the cost of GTP hydrolysis .During each translocation event, a new  charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal. Ribosomal movements  are induced by conformational changes that advance the ribosome by three bases in the 3′ direction.
• This process of elongation continues until the ribosome arrives at the stop codon.
• Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds.

Termination

• Termination occurs when the ribosome encounters a stop codon UAG/UAA and UGA/UAA, respectively in the mRNA.
• These stop codons are recognized by the termination (or release) factors RF1 and RF2.
• Another termination factor, RF3, facilitates turnover of RF1 and RF2 but is not required for peptidyl-tRNA hydrolysis.
• The mechanism of termination encompasses  three steps:
  • recognition of the stop codon,
  • hydrolysis of the ester bond of the peptidyl-tRNA (these two steps are accomplished by RF1 or RF2) and
  • dissociation of RF1/RF2 with the help of RF3.

"Translation in bacteria" by Biology 2e. Provided by: OpenStax is licensed under CC BY 4.0

Bacteria such as E. coli need amino acids  like tryptophan to survive, which they can ingest from the environment.

Histidine-Trpoperon by Wikimedia Commons licensed under CC BY-SA 3.0

Attenuation of the trp Operon

• The leader sequence (L) of the trp Operon lies before the trpE gene at its 5' end . This sequence about 160 bp is size and controls the expression of the operon through a process called attentuation.
• This sequence has four domains (1-4). Domain 3 (nucleotides 108-121) of the mRNA can base pair with either domain 2 (nucleotides 74-94) or domain 4 (nucleotides 126-134).
•  At high levels of tryptophan in the medium :  domain 3 pairs with domain 4, a stem and loop structure forms on the mRNA and transcription stops.
• At low levels of tryptophan in the medium :     domain 3 pairs with domain 2, then the stem and loop structure does not form and transcription continues through the operon, and all of the enzymes required for tryptophan biosynthesis are produced.
• Attenuators: Domain 4 is called the attenuator because its presence is required to reduce (attenuate) mRNA transcription in the presence of high levels of tryptophan.
• Domain 1 is also an important component of the attenuation process. The section of the leader sequence encodes a 14 amino acid peptide that has two tryptophan residues.

• When the cellular levels of tryptophan are high, the levels of the tryptophan tRNA are also high.
• Immediately after transcription, the mRNA moves quickly through the ribosome complex translating the leader peptide .
• Translation is quick because of the high levels of tryptophan tRNA.
• Domain 2 becomes associated with the ribosome complex.
• As result the domain 3 binds with domain 4, and
• transcription is attenuated because of the stem and loop formation.

• Under low cellular levels of tryptophan, the translation of the peptide on domain 1 is slow.
• Domain 2 does not become associated with the ribosome.
• Rather domain 2 associates with domain 3.
• This structure permits the continued transcription of the operon.
• Then the trpE-A genes are translated, and the biosynthesis of tryptophan occurs.

Trp operon attenuation by Wikimedia commons is licensed under CC BY-SA 3.0

 RNA polymerase II requires specific proteins called transcription factors to initiate transcription . 

Two general types of transcription factors have been defined  namely ;

• General transcription factors  : These are involved in transcription from all polymerase II promoters and  part of the basic transcription machinery.
• Additional transcription factors  : These bind to DNA  sequences that control the expression of individual genes and  regulate gene expression.

Enhancers

Enhancer regions are binding sequences,  for specific transcription factors. [pb_glossary id="934"]Enhancers[/pb_glossary] may be located upstream or downstream of transcription start site. Each enhancer is made up of short DNA sequences called distal control elements .

Proteins called Activators  , mediator proteins and transcription factors bind to the enhancers .When a transcription factor binds to its enhancer sequence, the shape of the  transcription factor protein changes. This enables the transcription factor to   interact with proteins at the promoter site.

As the enhancer region may be distant from the promoter, the DNA must bend to allow the proteins at the two sites to come into contact. DNA bending proteins help to bend the DNA and bring the enhancer and promoter regions together.

 Two different genes may have the same promoter but different distal control elements, enabling differential gene expression.

"Enhancers" image from Biology, an OpenStax resource is licensed under CC BY 4.0

The promoters of RNA polymerase II

 The TATA box : This resembles the the -10 sequence element of bacterial promoters. This region contain the sequence TATAA  and is located 25 to 30 nucleotides upstream of the transcription start site.

Initiator, or Inr, sequence : In addition to the TATA box , the promoters of many gene contain a second important sequence element called initiator, or Inr, sequence. Some RNA polymerase II promoters contain only an Inr element, with no TATA box. 

The promoter -proximal elements : The CAAT box, with the consensus sequence 5’-CCAAT-3’ and the GC box, with the consensus sequence 5’-GGGCGG-3’. These  promoter-proximal elements  regulate gene transcription.

Initiation-Formation of the Transcription complex

• The first step in formation of a transcription complex is the binding of a general transcription factors called TFIID to the TATA box.TFIID is itself composed of multiple subunits,  including the TATA binding protein . This binds specifically to the TATAA consensus sequence .and 10-12 other polypeptides, called TBP-associated factors (TAFs).
• TBP then binds a second general transcription factor (TFIIB) forming a TBP-TFIIB complex at the along with TF II F at the promoter
• Then RNA polymerase II binds
• This is followed by the binding of TFIIE and TFIIH.
• TFIIH is a multi subunit factor. It plays  two important roles. First, two subunits of TFIIH are helicases, which unwind DNA  around the initiation site. These subunits of TFIIH are also required for Nucleotide excision repair .Another subunit of TFIIH is a  Protein kinase . This phosphorylates repeated sequences present in the C-terminal domain of the largest subunit of RNA polymerase II. 

"Eukaryotic Transcription Initiation" image from the book General Biology (Boundless) licensed under CC BY-SA 4.0

• Once the complex is assembled, RNA polymerase can bind to its upstream sequence.
• When bound along with the transcription factors, RNA polymerase is phosphorylated. This releases part of the protein from the DNA .  The transcription initiation complex  gets activated and places RNA polymerase in the correct orientation to begin transcription.
• Then the double-stranded DNA in the transcription start region  is unwound . The RNA Polymerase II is then  positioned at the +1 initiation nucleotide and starts new RNA strand synthesis.

The promoters recognized by RNA polymerase I consists of

•  a core element  :  It  surrounds the transcription start site , and
• an upstream promoter element  :  This  is about 100 bp farther upstream.
• RNA polymerase  binds to promoter containing a core promoter element and an upstream control element (UCE). The Tata Binding Protein ( TBP), which is part of a larger complex called SL1, helps RNA polymerase I to recognize the core promoter

The promoters of RNA polymerase III

• The RNA polymerase III promoters are of two types namely  Type I and Type II . These  promoters  lie wholly within the genes. 
• The type II promoter of the Xenopus laevis 5S RNA gene consists of an internal control region (ICR). This is  subdivided into A box (+50 to +60), intermediate element (IE, +67 to +72), and C box (+80 to +90).
• The type II  promoter of the X. laevis tRNA^Leugene consists of an A box (+8 to +19) and a B box (+52 to +62).
• The type III promoter of the Homo sapiens U6 snRNA gene consists of a distal sequence element (DSE, −215 to −240)  and a core promoter composed of a proximal sequence element (PSE, −65 to −48) and a TATA box (−32 to −25). 

Elongation

• RNA Polymerase  clears or “escapes” the promoter region and leaves most of the transcription initiation proteins behind.

• RNA Polymerases travel along the template DNA strand in the 3′ to 5′ direction .The synthesis of new RNA strands takes place in the 5′ to 3′ direction. i.e.,  new nucleotides are added to the 3′ end of the growing RNA strand.

• RNA Polymerases unwind the double stranded DNA ahead of them and rewinds the unwound DNA behind them.
• RNA strand synthesis occurs in a transcription bubble of about 25 unwound DNA base pairs.
• About 8 nucleotides of newly-synthesized RNA remain base paired to the template DNA. The rest of the RNA molecules falls off the template  and this  allows the DNA behind it to rewind.

Termination

• The termination of transcription is different for the three different eukaryotic RNA polymerases.
• RNA Polymerase I contain a specific sequence of base pairs -11 bp long in humans  :  18 bp in mice .  This sequence is recognized by a termination protein called TTF-1 (Transcription Termination Factor for RNA Polymerase I.).  This protein binds the DNA at its recognition sequence and blocks further transcription, causing the RNA Polymerase I to detach from the template DNA strand and  releases the newly-synthesized RNA.
• RNA Polymerse II lack any specific signals or sequences that direct RNA Polymerase II to terminate at specific locations. The transcript is cleaved at an internal site before RNA Polymerase II finishes transcribing. The cleavage site occurs between an upstream AAUAAA sequence and a downstream GU-rich sequence separated by about 40-60 nucleotides in the emerging RNA.
•  The upstream portion of the transcript is released .
• The remainder of the transcript is digested by a 5′-exonuclease (called Xrn2 in humans) while it is still being transcribed by the RNA Polymerase II. When the 5′-exonulease “catches up” to RNA Polymerase II by digesting away all the overhanging RNA, it  disengage the polymerase from its DNA template strand, finally terminating transcription.
• A protein called CPSF in humans binds the AAUAAA sequence and a protein called CstF in humans binds the GU-rich sequence. CPSF cleaves the nascent pre-mRNA at a site 10-30 nucleotides downstream from the AAUAAA site. The Poly(A) Polymerase enzyme  catalyze the addition of a 3′ poly-A tail on the pre-mRNA .
• The RNAs transcribed by RNA Polymerase III have a short stretch of four to seven U’s at their 3′ end. This somehow triggers RNA Polymerase III to both release the nascent RNA and disengage from the template DNA strand.

• Translation is the last step in gene expression,
• During translation  the coding sequence of mRNA is translated into the amino-acid sequence of a protein.
• Translation is a highly dynamic process and comprises  four major phases: initiation, elongation, termination, and ribosome recycling.
• Ribosomes form transient complexes with auxiliary translation factors during each phase of translation and  facilitate protein synthesis. 

Translation in Prokaryotes

• In bacteria, translation initiation occurs co transcriptionally. The RNA polymerase (RNAP) and the ribosome physically interact with each other.
• The ribosome binds to the ribosome binding site (RBS) of the mRNA as soon as it emerges from the RNAP.

Initiation

• During translation initiation, the ribosome recruits an mRNA and selects the start codon of the Open Reading Frame (ORF) 
• m RNAS of prokaryotes have an extended 5′ untranslated region (5′UTR) and an [pb_glossary id="936"]Shine- Dalgarno sequence [/pb_glossary](SD) located 8–10 nt upstream of the start codon (usually AUG).
• Interactions between the SD sequence and the complementary anti-SD (aSD) sequence in 16S ribosomal RNA (rRNA) recruits the small 30 S ribosomal subunit and anchors  the 30S ribosomal subunit at the correct location on the mRNA template.
• Initiation is promoted by initiation factors IF1, IF2, and IF3. IF1 enhances the activities of IF2 and IF3.IF2 is a GTPase that recruits the initiator fMet-tRNA^fMet. IF3 interferes with subunit association. It also ensures the fidelity of fMet-tRNA^fMet selection over the elongator aminoacyl-tRNAs (aa-tRNAs), and helps to discriminate against mRNAs with unfavorable translation initiation regions (TIRs)
• The initiator tRNA then interacts with the start codon AUG (or rarely, GUG). This tRNA carries the amino acid methionine, which is formylated after its attachment to the tRNA and thus the  Charged t RNA , fMet-tRNA^Metf    is formed .  This is mediated by the initiation factor IF-2.
• The initiating fMet is usually removed after translation is complete.
• After the formation of the initiation complex, the 30S ribosomal subunit is joined by the 50S subunit to form the 70S translation complex. 

"Prokaryotic Translation Initiation" by Richard Wheeler (Zephyris) via wikimedia is licensed under CC BY-SA 3.0

• The ribosome consists of three compartments.
  • The A (aminoacyl) site  : the incoming charged aminoacyl tRNAs binds to this site.
  • The P (peptidyl) site  : binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA.
  • The E (exit) site  :  The uncharged t RNA  ( t RNA after delivering the aminoacid) are released from this site.

Differences between Prokaryotic and Eukaryotic Translation 

• Ribosome dissociation in to 40 S and 60 S
• Ternary complex  called pre initiation complex formation – initiator t RNA +GTP+ eIF2+40S ribosomal subunit
• mRNA binding to preinitiation complex
• 60S ribosomal subunit binding to the complex

The steps in the process of initiation

• GTP binds to eIF2- Binary complex
• eIF2 composed of 3 subunits – α, β and γ.
• Binary complex binding to initiator t RNA
• Binding of 40S ribosomal subunit
• Ternary complex – called 43S preinitiation complex.  This complex is stabilized by earlier  association of eIF3 and eIF1 to the 40S subunit
• Eukaryotes does not require the Shine-Dalgarno sequence rather the eukaryotic initiation complex recognizes the 7-methylguanosine cap at the 5' end of the mRNA.
• Cap structure of mRNA is bound by eIFs prior to pre initiation complex formation. Cap binding accomplished by e IF -4F
  This factor is a complex of 3 proteins  namely  eIF-4E, A and G 
  eIF-4E    -    24 KDa protein, recognizes and binds cap structure
  eIF-4A   -    46 KDa protein, binds and hydrolyses ATP , exhibits RNA helicase activity, resolves RNA secondary structures.
  eIF- 4G    - helps in the binding of mRNA to 43S preinitiation complex
• The cap-binding protein (CBP) and  IFs assist the movement of the ribosome to the 5' cap. Once at the cap, the initiation complex moves  along the mRNA in the 5' to 3' direction, searching for the AUG start codon.
• Many eukaryotic mRNAs are translated from the first AUG, but this is not always the case.
• According to Kozak’s rules, the nucleotides around the AUG indicate whether it is the correct start codon. [pb_glossary id="938"]Kozak’s rules[/pb_glossary] state that the following consensus sequence must appear around the AUG of vertebrate genes: 5'-gccRccAUGG-3'. The R (for purine) indicates a site that can be either A or G, but cannot be C or U. Sequences closer to this consensus show higher efficiency of translation.
• e IF-5 binds to preinitiation complex. This is followed by the binding of initiator t RNA – met –tRNA met   to the AUG codon of mRNA ( process helped by eIF-1)
• This is followed by the binding of 60S subunit and results in the formation of 80S complex.
• Association of 60S requires e IF-5.Energy for the binding dervied by GTP hydrolysis – bound to eIF-2.

• eIF-2 cycle

Regulation

Termination

GENE REGULATION

• • https://www.nature.com/scitable/topicpage/gene-expression-14121669/
  • https://iu.pressbooks.pub/iul211smehta/chapter/regulation-of-transcription-in-prokaryotes/
  • https://courses.lumenlearning.com/suny-wmopen-biology1/chapter/outcome-prokaryotic-gene-regulation/
   

Gene expression regulation enables the human body to respond to changes in nutrient concentration

Gene expression regulation helps to conserve Energy and Space 

• Chromatin accessibility. The structure of chromatin can be regulated. More open or “relaxed” chromatin makes a gene more available for transcription.
• Transcription. Transcription is a key regulatory point for many genes. With the help of transcription factor proteins bind to specific DNA sequences in or near a gene and promote or repress its transcription into an RNA.
• RNA processing. Processing events like Splicing, capping, alternative splicing and addition of a poly-A tail to an RNA molecule can be regulated in turn regulating gene expression.
• RNA stability. The lifetime of an mRNA molecule in the cytosol determines the number of proteins can be made from it. Small regulatory RNAs called miRNAs can bind to target mRNAs and cause them to be chopped up.
• Translation. Translation of an mRNA may be increased or inhibited by regulators. For instance, miRNAs sometimes block translation of their target mRNAs (rather than causing them to be chopped up).
• Protein activity. Proteins can undergo a variety of modifications, such as being chopped up or tagged with chemical groups. These modifications can be regulated and this in turn affect the activity or behavior of the protein.

This  Open textbook on "Understanding Gene Regulation and Gene expression" developed by Dr. V. Malathi as part of her OE4BW project in 2023.

This book will be an essential part of courses in Molecular Biology, Genetics , Genetic Engineering and Biotechnology . This project is aimed at creating an open textbook for the global student community. This open text book will discuss the following topics along with external links for further reading.

• The organization of genes in Prokaryotes and eukaryotes
• Types of chromosomes
• The process of gene expression, Transcription and translation in eukaryotes & prokaryotes
• The stages at which gene expression can be regulated
• The importance of gene expression and its regulation

These  topics are described using a simple language with suitable illustrations, interactive exercises (created using H5P).

The content  includes reading/watching materials adapted from openly licensed OERs from open repositories like OpenStax, Lumen Learning, Libre Texts , which are  suitably re-used, re-purposed, adapted with proper attribution.

"cAMP binds to the CAP protein, a positive regulator" from Biology, an OpenStax resource is licensed under CC BY-SA 4.0

• Predict the effects on lactose metabolism when the various genes and DNA control elements are mutated (added or removed).
• Predict the effects on lactose metabolism when the concentration of lactose is changed.
• Explain the roles of LacI, LacZ, and LacY in lactose regulation.

• Explain the main sequence of events that occur within a cell that leads to protein synthesis.
• Predict how changing the concentrations and interactions of biomolecules affects protein production.
• Explain how protein production in a single cell relates to the quantity produced by a collection of cells.

"Gene Expression Essentials " by PHET is licensed under CC BY 4.0

cis-acting DNA element

cis-acting DNA elements- enhancers and silencers

Trans-acting DNA element

Post-Translational Control of Gene Expression

• RNA is transcribed, but must be processed into a mature form before translation can begin. This processing is called post-transcriptional modification. 
• This post-transcriptional step can also be regulated to control gene expression in the cell. 
• In eukaryotic  RNA transcript often contains regions, called introns, that are removed prior to translation.
• The regions of RNA that code for protein are called exons .
• By a process called[pb_glossary id="948"] splicing[/pb_glossary] , the RNA is processed and the introns are removed and exons are ligated together.

"Pre-mRNA can be alternatively spliced to create different proteins" by Shelli Carter and Lumen Learning. is licensed under CC BY 4.0

ALTERNATIVE RNA SPLICING

• Alternative RNA splicing is a mechanism that allows different protein products to be produced from one gene when different combinations of introns, and sometimes exons, are removed from the transcript.
• Alternative splicing  acts as a mechanism of gene regulation,
• The frequency of different splicing alternatives controlled by the cell as a way to control the production of different protein products in different cells or at different stages of development.
•  70 percent of genes in humans are expressed as multiple proteins through alternative splicing.

"Splicing_overview.jpg" by Agathman is licensed under CC BY-SA 3.0

Control of RNA Stability

•  The  3' and 5' ends of the mRNA is protected before it  leaves the nucleus,
• .The 5′ end of the mRNA is protected by a  5′ cap, which is usually composed of a methylated guanosine triphosphate molecule (GTP).
• The poly-A tail protects the 3′ end. It is usually composed of a series of adenine nucleotides.
• The length of time that the RNA resides in the cytoplasm  can be controlled.
• Each RNA molecule has a defined lifespan and decays at a specific rate. This rate of decay determine how much protein is in the cell. For example  , If the decay rate is increased, the RNA will not exist in the cytoplasm as long, shortening the time for translation to occur.
• This rate of decay is referred to as the RNA stability
• Proteins, called RNA-binding proteins, or RBPs, can bind to the regions of the RNA just upstream or downstream of the protein-coding region. These regions in the RNA that are not translated into protein are called the [pb_glossary id="950"]untranslated regions, or UTRs.[/pb_glossary]
• These regions  regulate mRNA localization, stability, and protein translation. The region just before the protein-coding region is called the 5′ UTR, whereas the region after the coding region is called the 3′ UTR .
• The binding of RBPs to these regions can increase or decrease the stability of an RNA molecule, depending on the specific RBP that binds.

"The protein-coding region of mRNA is flanked by 5′ and 3′ untranslated regions (UTRs). The presence of RNA-binding proteins at the 5′ or 3′ UTR influences the stability of the RNA molecule." by Shelli Carter and Lumen Learning is licensed under CC BY 3.0

 microRNAs

•  microRNAs, or miRNAs, are short RNA molecules that are only 21–24 nucleotides in length.
• The miRNAs are made in the nucleus as longer pre-miRNAs.
• A protein called dicer chop these pre-miRNAs are chopped into mature miRNAs . 
• Mature miRNAs recognize a specific sequence and bind to the RNA and associate with a ribonucleoprotein complex called the RNA-induced silencing complex (RISC).
• RISC binds along with the miRNA to degrade the target mRNA. Together, miRNAs and the RISC complex rapidly destroy the RNA molecule.

Role of Histones in gene regulation

Histone Modifications

• • methylation,
  • phosphorylation,
  • acetylation,
  • ubiquitylation, and
  • sumoylation. (small ubiquitin-related modifier (SUMO) family are conjugated to proteins to regulate such cellular processes as nuclear transport, transcription, chromosome segregation and DNA repair.)
• Histones modifications is  carried out by acetyltransferases and deacetylases, methyltransferases and demethylases.
• The PTMs made to histones can impact gene expression by altering chromatin structure or recruiting histone modifiers.

Histone -Lysine acetylation

• Histone -Lysine acetylation is regulated by two enzymes:
• Histone Acetyltransferases (HATs)–use acetyl-CoA that is specifically recognized and bound by the Arg/Gln-X-X-Gly-X-Gly/Ala segment of HATs to transfer an acetyl group to the ε-amino groups on the N-terminal tails of histones .
• Histone Deacetylases (HDACs)–reverses the above modification by HATs
• Lysine acetylation causes a destabilization of the nucleosome and chromatin structure–effectively facilitating access to the DNA for various nuclear factors like the transcription complex.
• Hyperacetylated histones are regarded to be a hallmark of transcriptionally active chromatin.
• The work of HDACs increases the affinity between the nucleosome and the DNA, leading to a closed (heterochromatin-like) chromatin conformation that minimizes accessibility for the transcriptome.

Histone methylation

• This unique posttranslational modification is performed by a specific enzyme family known as the Protein Arginine N-Methyltransferases (PRMTs).
• Along with serine/threonine phosphorylation, and lysine methylation and acetylation, arginine methylation is an epigenetic histone modification that regulates gene expression as part of the histone code

Chromatin remodeling

Go through the presentation given below and answer the questions.

 Altered Gene Expression and Cancer

Silencing genes through epigenetic mechanisms is also very common in cancer cells. Certain histone and DNA  modifications are associated with silenced genes. The DNA in the promoter region of silenced genes is methylated on cytosine DNA residues in CpG islands in most cancer cells.

[pb_glossary id="958"]Systemic Lupus Erythematosus (SLE)[/pb_glossary]

Psoriasis

[pb_glossary id="962"]Rheumatoid Arthritis  (RA)[/pb_glossary]

[pb_glossary id="964"]Systemic Sclerosis (SSc) [/pb_glossary]

SSc is a relatively rare disease which is characterized by damages of connective tissues mediated by autoreactive immune cells. Its etiopathogenesis remains unclear. Abnormal epigenetic modifications have been shown in SSc. Dysregulated DNA methylation is indicated in Systemic Sclerosis.

Very similar to Print My Blog Quick Print, but supporting Pro features. Great for making a PDF for an easy-to-read digital backup of your favorite website content.

Features

• Supports nesting articles (like posts and pages) into “parts”
• External hyperlinks (links to web pages not in the project) can be automatically converted to footnotes, left in-place, or removed
• Internal hyperlinks (links to content included in the project) can be automatically converted to to footnotes, inline page references, left in-place, or removed
• The default title page can include: Project Title, Subtitle, Site URL, Date Printed, Credit to Print My Blog
• Each article can include: Title, ID, Author, URL, Date Published, Categories and Tags, Featured Image, Excerpt, Custom Fields, Content
• Limit image size by setting maximum height
• Optionally automatically center images
• Optionally float images to the top or bottom of each page

Page Layout

• Page numbers in bottom-right corner, article titles in the top-right, and part titles (if using parts) appear in the top-left corner. Front-matter’s pages are numbered with Roman numerals
• Each article can optionally begin on a new page

Perfect for editing and reviewing your content! Compact to conserve paper, lots of meta-information, and double-spaced text to allow for editor’s notes.

Features

• Supports nesting articles (like posts and pages) into “parts”
• External hyperlinks (links to web pages not in the project) can be automatically converted to footnotes or removed
• Internal hyperlinks (links to content included in the project) can be automatically converted to footnotes, inline page references, or removed
• The default title page can include: Project Title, Subtitle, Site URL, Date Printed, Credit to Print My Blog
• Each article can include: Title, ID, Author, URL, Date Published, Categories and Tags, Featured Image, Excerpt, Custom Fields, Content
• Optionally automatically center images
• Optionally float images to the top or bottom of each page

Page Layout

This design is meant for printing on both sides of the page so you see two pages at once (the "page spread"). So the left-hand page will be styled differently than the right-hand page.

• Page numbers in bottom-outside corner, part titles appear in the top of the left page, article titles appear at the top of the right page. Front-matter’s pages are numbered with Roman numerals
• Each article can optionally begin on a new page

Very similar to Print My Blog Quick Print, but supporting Pro features. Great for printing your WordPress content.

Features

• Supports nesting articles (like posts and pages) into “parts”
• External hyperlinks (links to web pages not in the project) can be automatically converted to footnotes or removed
• Internal hyperlinks (links to content included in the project) can be automatically converted to footnotes, inline page references, or removed
• The default title page can include: Project Title, Subtitle, Site URL, Date Printed, Credit to Print My Blog
• Each article can include: Title, ID, Author, URL, Date Published, Categories and Tags, Featured Image, Excerpt, Custom Fields, Content
• Optionally automatically center images
• Optionally float images to the top or bottom of each page

Page Layout

This design is meant for printing on both sides of the page so you see two pages at once (the "page spread"). So the left-hand page will be styled differently than the right-hand page.

• Page numbers in bottom-outside corner, part titles appear in the top of the left page, article titles appear at the top of the right page. Front-matter’s pages are numbered with Roman numerals
• Each article can optionally begin on a new page

For creating a printout of your WordPress content that saves paper and ink.

This design uses a smaller font, small images, doesn’t add pagebreaks between articles, and avoids unnecessary extra information.

Features

• Supports nesting articles (like posts and pages) into “parts”
• External hyperlinks (links to web pages not in the project) can be automatically converted to footnotes or removed
• Internal hyperlinks (links to content included in the project) can be automatically converted to footnotes, inline page references, or removed
• The default title page can include: Project Title, Subtitle, Site URL, Date Printed, Credit to Print My Blog
• Each article can include: Title, ID, Author, URL, Date Published, Categories and Tags, Featured Image, Excerpt, Custom Fields, Content
• Optionally automatically center images
• Optionally float images to the top or bottom of each page

Page Layout

This design is meant for printing on both sides of the page so you see two pages at once (the "page spread"). So the left-hand page will be styled differently than the right-hand page.

• Page numbers in bottom-outside corner, part titles appear in the top of the left page, article titles appear at the top of the right page. Front-matter’s pages are numbered with Roman numerals
• Each article can optionally begin on a new page

Designed for a school, company, or organization that wants to include their name, logo, and colors throughout the PDF. Because of its exstensive use of colors, it’s usually best viewed from a computer or handheld device.

Features

• Easier content editing because there are no nesting parts.
• All hyperlinks converted into footnotes
• The title page can include your organization’s name, project title, issue number, list of authors, and an introductory paragraph.
• Each article includes just its title and content

Page Layout

• The default title page includes a stylized banner (with customizable color) above a colored background featuring a large version of your company logo.
• Main matter pages fade from white to a color of your choosing in the corner where your logo and page number are shown.
• Page numbers in bottom-right corner, and article titles in the top-right. Front-matter’s pages are numbered with Roman numerals
• Each article begins on a new page
• Back matter uses your large company logo with background color

Designed to look good when viewed from a device or printing to paper. The two-column layout leads to more compact content, especially good if your content doesn’t require the full page width

Features

• Main matter is divided into two columns, while front matter uses the full page width.
• Content can be divided into parts with a special part opening area using the full page width.
• The title page can include a title and preamble. Other content can immediately follow, without needing a page break.
• Each article includes just its title and content, and an optional dividing line to help distinguish between articles.
• If your content is already divided into columns, optionally automatically remove them to improve the layout.
• Optionally begin each post on a new page, or be shown immediately following the previous one.
• Optionally have each article title take up the full page width, or show them inside columns.
• Internal links (hyperlinks to content included in the project) are replaced with page references.
• External links (hyperlinks to content on the internet) are left as-is.

Page Layout

• Most content is divided between two columns.
• Page numbers in bottom-right corner, and part titles in the top-left. Front-matter’s pages are numbered with Roman numerals

Special Instructions

Front matter defaults to using the full page width. To force something to be divided into columns, add the CSS class "mayer-columns" inside your content.

Main matter defaults to being divided into columns. To force something to use the full page width, add the CSS class "mayer-wide" inside the content.

Similar to Print My Blog Quick Print and dotEpub, but supporting Pro features. Great for printing your WordPress content as an eBook for reading from a tablet or phone, or uploading to eBook marketplaces like Amazon or Apple Book Store.

A table of contents is generated and inserted into the book (no need to add it using an external service like Kindle Direct Publishing or Calibre).

Images are bundled into the book file so they can be used offline.

Image galleries are rearranged into a simple list of images which displays better on smaller devices.

If there are any features or options you want, please get in touch.

Features

• Supports nesting articles (like posts and pages) into “parts”
• External hyperlinks (links to web pages not in the project) are left as working hyperlinks
• Internal hyperlinks (links to content included in the project) are converted into links to the appropriate page of the eBook
• The default title page can include: Project Title, Subtitle, Site URL, Date Printed, Credit to Print My Blog
• Each article can include: Title, ID, Author, URL, Date Published, Categories and Tags, Featured Image, Excerpt, Custom Fields, Content
• All images are automatically included in the ePub file (instead of merely pointing to the external image source). This makes the filesize larger, but allows users to view images while disconnected from the Internet.
• Small images are automatically centered
• Image galleries are converted into regular list of images, which look better on small screens.

Page Layout

The page design is mostly dictated by the app and device used for reading

Each article begins on a new page.

Similar to Print My Blog Quick Print's other "Classic" designs, except optimized for reading by Microsoft Word. Useful when you need to deliver your content as a Microsoft Word Document (like to an editor), or for using as an intermediate format for delivery to other applications (like for importing into Adobe InDesign).

A table of contents is generated and inserted at the requested location. Once in Microsoft Word, right click on the table of contents and click "Update Field"; or press Ctrl + A, then F9.

Images may be bundled into the Word Document file so they can be used offline or if the website hosting the images goes offline.

Image galleries are rearranged into a simple list of images which displays better on smaller devices.

If there are any features or options you want, please get in touch.

Features

• Supports nesting articles (like posts and pages) into “parts”
• External hyperlinks (links to web pages not in the project) may be left as working hyperlinks or removed
• Internal hyperlinks (links to content included in the project) may be converted into links to the appropriate page of the document, left as hyperlinks to the website, or removed.
• The default title page can include: Project Title, Subtitle, Site URL, Date Printed, Credit to Print My Blog
• Each article can include: Title, ID, Author, URL, Date Published, Categories and Tags, Featured Image, Excerpt, Custom Fields, Content
• All images are automatically included in the Word Document file (instead of merely pointing to the external image source). This makes the filesize larger, but allows users to view images while disconnected from the Internet.
• Small images are automatically centered
• Image galleries are converted into regular list of images, which look better on small screens.

Page Layout

Each article begins on a new page.

Multi-column newspaper design designed for printing. Wide images can span multiple columns and always snap to the nearest page edge to make a visually-appealing layout.

Features

• The "Title page" is actually a special header on the front page containing your publication's title, issue number, date, and other optional prominent info (set on the design and project meta settings).
• Content is divided into two, three, or four columns.
• The top margin contains the front page (publication title, issue number, date, etc.).
• Designed for duplex printing with the left page mirroring the right page's margins.
• Content can be divided into parts. Part openings and important articles' titles and cover images take up the full page width, whereas regular articles compactly fit into columns.
• Each article may include post title, ID, author, published date, categories and tags, URL, featured image, excerpt, custom fields, and content.
• If your content is already divided into columns, optionally automatically remove them to improve the layout.
• External hyperlinks (links to web pages not in the project) can be automatically converted to footnotes or removed
• Internal hyperlinks (links to content included in the project) can be automatically converted to footnotes, inline page references, or removed

Page Layout

• Issue and page number are in the outside top margin, the publication's title is in the middle top margin, and the publication date is in the inside top margin. The optional publication subtitle is underneath the other top-margin content.

Section Templates

Choose different styles for articles included in your project:

• Default Template: shows all content specified by the "Post Content" design setting (e.g. the article's title and featured image) in columns.
• Single Column: like the default template, but in a single column.
• Fullpage Content: contains only the article's content (no title, featured image, etc.) all in a single, wide column.
• Full Column Content: like "Fullpage Content", except content is still divided into multiple columns..
• Important: shows the article's featured image and title across all columns, but other content is shown in multiple columns.

Parts start on a new page and include the part’s title, featured image, and content.

Special Instructions

Note that in order to improve page layout, all images and figures snap to the nearest page edge.

"Full width" images take up all columns and snap to the nearest page edge.

"Wide width" images take up 2 columns and snap to the nearest page edge.

Add the CSS class "pmb-dont-snap" to any images you don't want to snap like this.