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12.14: pre-RNA and mRNA - Biology

12.14: pre-RNA and mRNA - Biology


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After transcription, eukaryotic pre-mRNAs must undergo several processing steps before they can be translated. Eukaryotic (and prokaryotic) tRNAs and rRNAs also undergo processing before they can function as components in the protein synthesis machinery.

MRNA Processing

The eukaryotic pre-mRNA undergoes extensive processing before it is ready to be translated. The additional steps involved in eukaryotic mRNA maturation create a molecule with a much longer half-life than a prokaryotic mRNA. Eukaryotic mRNAs last for several hours, whereas the typical E. coli mRNA lasts no more than five seconds.

Pre-mRNAs are first coated in RNA-stabilizing proteins; these protect the pre-mRNA from degradation while it is processed and exported out of the nucleus. The three most important steps of pre-mRNA processing are the addition of stabilizing and signaling factors at the 5′ and 3′ ends of the molecule, and the removal of intervening sequences that do not specify the appropriate amino acids. In rare cases, the mRNA transcript can be “edited” after it is transcribed.

5′ Capping

While the pre-mRNA is still being synthesized, a 7-methylguanosine cap is added to the 5′ end of the growing transcript by a phosphate linkage. This moiety (functional group) protects the nascent mRNA from degradation. In addition, factors involved in protein synthesis recognize the cap to help initiate translation by ribosomes.

3′ Poly-A Tail

Once elongation is complete, the pre-mRNA is cleaved by an endonuclease between an AAUAAA consensus sequence and a GU-rich sequence, leaving the AAUAAA sequence on the pre-mRNA. An enzyme called poly-A polymerase then adds a string of approximately 200 A residues, called the poly-A tail. This modification further protects the pre-mRNA from degradation and signals the export of the cellular factors that the transcript needs to the cytoplasm.

Pre-mRNA Splicing

Eukaryotic genes are composed of exons, which correspond to protein-coding sequences (ex-on signifies that they are expressed), and intervening sequences called introns (intron denotes their intervening role), which may be involved in gene regulation but are removed from the pre-mRNA during processing. Intron sequences in mRNA do not encode functional proteins.

The discovery of introns came as a surprise to researchers in the 1970s who expected that pre-mRNAs would specify protein sequences without further processing, as they had observed in prokaryotes. The genes of higher eukaryotes very often contain one or more introns. These regions may correspond to regulatory sequences; however, the biological significance of having many introns or having very long introns in a gene is unclear. It is possible that introns slow down gene expression because it takes longer to transcribe pre-mRNAs with lots of introns. Alternatively, introns may be nonfunctional sequence remnants left over from the fusion of ancient genes throughout evolution. This is supported by the fact that separate exons often encode separate protein subunits or domains. For the most part, the sequences of introns can be mutated without ultimately affecting the protein product.

All of a pre-mRNA’s introns must be completely and precisely removed before protein synthesis. If the process errs by even a single nucleotide, the reading frame of the rejoined exons would shift, and the resulting protein would be dysfunctional. The process of removing introns and reconnecting exons is called splicing (Figure 1). Introns are removed and degraded while the pre-mRNA is still in the nucleus. Splicing occurs by a sequence-specific mechanism that ensures introns will be removed and exons rejoined with the accuracy and precision of a single nucleotide. The splicing of pre-mRNAs is conducted by complexes of proteins and RNA molecules called spliceosomes.

Practice Question

Errors in splicing are implicated in cancers and other human diseases. What kinds of mutations might lead to splicing errors?

[practice-area rows=”2″][/practice-area]
[reveal-answer q=”454729″]Show Answer[/reveal-answer]
[hidden-answer a=”454729″]Think of different possible outcomes if splicing errors occur. Mutations in the spliceosome recognition sequence at each end of the intron, or in the proteins and RNAs that make up the spliceosome, may impair splicing. Mutations may also add new spliceosome recognition sites. Splicing errors could lead to introns being retained in spliced RNA, exons being excised, or changes in the location of the splice site.[/hidden-answer]

Note that more than 70 individual introns can be present, and each has to undergo the process of splicing—in addition to 5′ capping and the addition of a poly-A tail—just to generate a single, translatable mRNA molecule.

Try It

The trypanosomes are a group of protozoa that include the pathogen Trypanosoma brucei, which causes sleeping sickness in humans (Figure 2). Trypanosomes, and virtually all other eukaryotes, have organelles called mitochondria that supply the cell with chemical energy. Mitochondria are organelles that express their own DNA and are believed to be the remnants of a symbiotic relationship between a eukaryote and an engulfed prokaryote. The mitochondrial DNA of trypanosomes exhibit an interesting exception to The Central Dogma: their pre-mRNAs do not have the correct information to specify a functional protein. Usually, this is because the mRNA is missing several U nucleotides. The cell performs an additional RNA processing step called RNA editing to remedy this.

Other genes in the mitochondrial genome encode 40- to 80-nucleotide guide RNAs. One or more of these molecules interacts by complementary base pairing with some of the nucleotides in the pre-mRNA transcript. However, the guide RNA has more A nucleotides than the pre-mRNA has U nucleotides to bind with. In these regions, the guide RNA loops out. The 3′ ends of guide RNAs have a long poly-U tail, and these U bases are inserted in regions of the pre-mRNA transcript at which the guide RNAs are looped. This process is entirely mediated by RNA molecules. That is, guide RNAs—rather than proteins—serve as the catalysts in RNA editing.

RNA editing is not just a phenomenon of trypanosomes. In the mitochondria of some plants, almost all pre-mRNAs are edited. RNA editing has also been identified in mammals such as rats, rabbits, and even humans. What could be the evolutionary reason for this additional step in pre-mRNA processing? One possibility is that the mitochondria, being remnants of ancient prokaryotes, have an equally ancient RNA-based method for regulating gene expression. In support of this hypothesis, edits made to pre-mRNAs differ depending on cellular conditions. Although speculative, the process of RNA editing may be a holdover from a primordial time when RNA molecules, instead of proteins, were responsible for catalyzing reactions.


The eukaryotic pre-mRNA undergoes extensive processing before it is ready to be translated. The additional steps involved in eukaryotic mRNA maturation create a molecule with a much longer half-life than a prokaryotic mRNA. Eukaryotic mRNAs last for several hours, whereas the typical E. coli mRNA lasts no more than five seconds.

The three most important steps of pre-mRNA processing are the addition of stabilizing and signaling factors at the 5′ and 3′ ends of the molecule, and the removal of intervening sequences that do not specify the appropriate amino acids.

5′ Capping

A cap is added to the 5′ end of the growing transcript by a phosphate linkage. This addition protects the mRNA from degradation. In addition, factors involved in protein synthesis recognize the cap to help initiate translation by ribosomes.

3′ Poly-A Tail

Once elongation is complete, an enzyme called poly-A polymerase adds a string of approximately 200 A residues, called the poly-A tail to the pre-mRNA. This modification further protects the pre-mRNA from degradation and signals the export of the cellular factors that the transcript needs to the cytoplasm.

Pre-mRNA Splicing

Eukaryotic genes are composed of exons, which correspond to protein-coding sequences (ex-on signifies that they are expressed), and intervening sequences called introns (intron denotes their intervening role), which are removed from the pre-mRNA during processing. Intron sequences in mRNA do not encode functional proteins.

All of a pre-mRNA’s introns must be completely and precisely removed before protein synthesis. If the process errs by even a single nucleotide, the reading frame of the rejoined exons would shift, and the resulting protein would be dysfunctional. The process of removing introns and reconnecting exons is called splicing (Figure 1).

Practice Question

Figure 1. Pre-mRNA splicing involves the precise removal of introns from the primary RNA transcript. The splicing process is catalyzed by protein complexes called spliceosomes that are composed of proteins and RNA molecules called snRNAs. Spliceosomes recognize sequences at the 5′ and 3′ end of the intron.

Errors in splicing are implicated in cancers and other human diseases. What kinds of mutations might lead to splicing errors?


The eukaryotic pre-mRNA undergoes extensive processing before it is ready to be translated. The additional steps involved in eukaryotic mRNA maturation create a molecule with a much longer half-life than a prokaryotic mRNA. Eukaryotic mRNAs last for several hours, whereas the typical E. coli mRNA lasts no more than five seconds.

Pre-mRNAs are first coated in RNA-stabilizing proteins these protect the pre-mRNA from degradation while it is processed and exported out of the nucleus. The three most important steps of pre-mRNA processing are the addition of stabilizing and signaling factors at the 5′ and 3′ ends of the molecule, and the removal of intervening sequences that do not specify the appropriate amino acids. In rare cases, the mRNA transcript can be “edited” after it is transcribed.

5′ Capping

While the pre-mRNA is still being synthesized, a 7-methylguanosine cap is added to the 5′ end of the growing transcript by a phosphate linkage. This moiety (functional group) protects the nascent mRNA from degradation. In addition, factors involved in protein synthesis recognize the cap to help initiate translation by ribosomes.

3′ Poly-A Tail

Once elongation is complete, the pre-mRNA is cleaved by an endonuclease between an AAUAAA consensus sequence and a GU-rich sequence, leaving the AAUAAA sequence on the pre-mRNA. An enzyme called poly-A polymerase then adds a string of approximately 200 A residues, called the poly-A tail. This modification further protects the pre-mRNA from degradation and signals the export of the cellular factors that the transcript needs to the cytoplasm.

Pre-mRNA Splicing

Eukaryotic genes are composed of exons, which correspond to protein-coding sequences (ex-on signifies that they are expressed), and intervening sequences called introns (intron denotes their intervening role), which may be involved in gene regulation but are removed from the pre-mRNA during processing. Intron sequences in mRNA do not encode functional proteins.

The discovery of introns came as a surprise to researchers in the 1970s who expected that pre-mRNAs would specify protein sequences without further processing, as they had observed in prokaryotes. The genes of higher eukaryotes very often contain one or more introns. These regions may correspond to regulatory sequences however, the biological significance of having many introns or having very long introns in a gene is unclear. It is possible that introns slow down gene expression because it takes longer to transcribe pre-mRNAs with lots of introns. Alternatively, introns may be nonfunctional sequence remnants left over from the fusion of ancient genes throughout evolution. This is supported by the fact that separate exons often encode separate protein subunits or domains. For the most part, the sequences of introns can be mutated without ultimately affecting the protein product.

All of a pre-mRNA’s introns must be completely and precisely removed before protein synthesis. If the process errs by even a single nucleotide, the reading frame of the rejoined exons would shift, and the resulting protein would be dysfunctional. The process of removing introns and reconnecting exons is called splicing (Figure 1). Introns are removed and degraded while the pre-mRNA is still in the nucleus. Splicing occurs by a sequence-specific mechanism that ensures introns will be removed and exons rejoined with the accuracy and precision of a single nucleotide. The splicing of pre-mRNAs is conducted by complexes of proteins and RNA molecules called spliceosomes.

Practice Question

Figure 1. Pre-mRNA splicing involves the precise removal of introns from the primary RNA transcript. The splicing process is catalyzed by protein complexes called spliceosomes that are composed of proteins and RNA molecules called snRNAs. Spliceosomes recognize sequences at the 5′ and 3′ end of the intron.

Errors in splicing are implicated in cancers and other human diseases. What kinds of mutations might lead to splicing errors?

Note that more than 70 individual introns can be present, and each has to undergo the process of splicing—in addition to 5′ capping and the addition of a poly-A tail—just to generate a single, translatable mRNA molecule.

RNA Editing in Trypanosomes

Figure 2. Trypanosoma brucei is the causative agent of sleeping sickness in humans. The mRNAs of this pathogen must be modified by the addition of nucleotides before protein synthesis can occur. (credit: modification of work by Torsten Ochsenreiter)

The trypanosomes are a group of protozoa that include the pathogen Trypanosoma brucei, which causes sleeping sickness in humans (Figure 2). Trypanosomes, and virtually all other eukaryotes, have organelles called mitochondria that supply the cell with chemical energy. Mitochondria are organelles that express their own DNA and are believed to be the remnants of a symbiotic relationship between a eukaryote and an engulfed prokaryote. The mitochondrial DNA of trypanosomes exhibit an interesting exception to The Central Dogma: their pre-mRNAs do not have the correct information to specify a functional protein. Usually, this is because the mRNA is missing several U nucleotides. The cell performs an additional RNA processing step called RNA editing to remedy this.

Other genes in the mitochondrial genome encode 40- to 80-nucleotide guide RNAs. One or more of these molecules interacts by complementary base pairing with some of the nucleotides in the pre-mRNA transcript. However, the guide RNA has more A nucleotides than the pre-mRNA has U nucleotides to bind with. In these regions, the guide RNA loops out. The 3′ ends of guide RNAs have a long poly-U tail, and these U bases are inserted in regions of the pre-mRNA transcript at which the guide RNAs are looped. This process is entirely mediated by RNA molecules. That is, guide RNAs—rather than proteins—serve as the catalysts in RNA editing.

RNA editing is not just a phenomenon of trypanosomes. In the mitochondria of some plants, almost all pre-mRNAs are edited. RNA editing has also been identified in mammals such as rats, rabbits, and even humans. What could be the evolutionary reason for this additional step in pre-mRNA processing? One possibility is that the mitochondria, being remnants of ancient prokaryotes, have an equally ancient RNA-based method for regulating gene expression. In support of this hypothesis, edits made to pre-mRNAs differ depending on cellular conditions. Although speculative, the process of RNA editing may be a holdover from a primordial time when RNA molecules, instead of proteins, were responsible for catalyzing reactions.


What is RNA?

Let&rsquos begin with the basics. Deoxyribonucleic acid (DNA) is a molecule you may already be familiar with it contains our genetic code, the blueprint of life. This essential molecule is the foundation for the &ldquocentral dogma of biology&rdquo, or the sequence of events necessary for life to function. DNA is a long, double-stranded molecule made up of bases, located in the cell&rsquos nucleus. The order of these bases determines the genetic blueprint, similar to the way the order of letters in the alphabet are used to form words. DNA&rsquos &lsquowords&rsquo are three letters (or bases) long, and these words specifically code for genes, which in the language of the cell, is the blueprint for proteins to be manufactured.

To &lsquoread&rsquo these blueprints, the double-helical DNA is unzipped to expose the individual strands and an enzyme translates them into a mobile, intermediate message, called ribonucleic acid (RNA). This intermediate message is called messenger RNA (mRNA), and it carries the instructions for making proteins. The mRNA is then transported outside of the nucleus, to the molecular machine responsible for manufacturing proteins, the ribosome. Here, the ribosome translates the mRNA using another three-letter word every three base pairs designates a specific building block called an amino acid (of which there are 20) to create a polypeptide chain that will eventually become a protein. The ribosome assembles a protein in three steps &ndash during initiation, the first step, transfer RNA (tRNA) brings the specific amino acid designated by the three-letter code to the ribosome. In the second step, elongation, each amino acid is sequentially connected by peptide bonds, forming a polypeptide chain. The order each amino acid is crucial to the functionality of the future protein errors in adding an amino acid can result in disease. Finally, during termination, the completed polypeptide chain is released from the ribosome and is folded into its final protein state. Proteins are required for the structure, function, and regulation of the body's tissues and organs their functionality is seemingly endless.

Throughout the latter half of the 20th century, we believed that RNA&rsquos primary role was to intermediate between DNA and protein, as we described above. Over the last three decades, those long-held beliefs have been shattered. We have witnessed amazing discoveries with regards to RNA biology, many of which have come from our own labs here at the RTI. In 1998, Andrew Fire and the RTI&rsquos Craig Mello discovered RNA interference (RNAi), in which double-stranded RNA can find and turn off specific genes based on certain sequences (order of the 'words'). For this, they earned the Nobel Prize in 2006! To understand more about RNAi and learn how we are developing this tool into a therapeutic platform, please see: What is RNAi?

This is an official Page of the University of Massachusetts Medical School

RNA Therapeutics Institute (RTI) &bull 368 Plantation St Worcester, Massachusetts 01605


Pre-mRNA is the primary transcript which contains both coding and non-coding sequences. mRNA is the mature messenger RNA which contains only the coding sequence of a gene. So, this is the key difference between pre-mRNA and mRNA. Pre-mRNA is subject to several processing steps, while mRNA is the product that results from the processing. Moreover, pre-mRNA does not travel to the cytoplasm while mRNA goes to the cytoplasm in order to produce a protein.

The below info-graphic tabulates more differences between pre-mRNA and mRNA.


Difference Between RNA and mRNA

Modern science says there are tiny building blocks which make up the blueprint of a human being’s genome. These micro-components control and decide the structure, function, and processes in every living cell. In the evolutionary period of life millions of years ago, the presence of these minute elements can trace us to where it all started and explain how life began to transform. Minute as they may be, these basic units have their own complexities. Considering two of them, they are the so-called RNA and mRNA.

RNA, or ribonucleic acid, is a chief and indispensable macromolecule (aside from DNA and proteins) of all kinds of existing life on the face of the Earth. RNA is also responsible in acting as a mediator in some of the biological processes of cells, such as directing genetic appearance, and communicating to the cell’s signals for a response. On the other hand, messenger RNA (mRNA) is a type or a particle of RNA also known as the “outline” for making protein. Messenger RNA is mainly in charge in the protein synthesis of the cell which is manufactured in the ribosome. Protein synthesis is the one accountable for the production of energy needed by the human body as well as the vital function of breathing thus, a very essential unit for survival.

RNA has three subtypes: mRNA, tRNA and rRNA. mRNA, also known as messenger RNA, is the key for the delivery of data from the structural gene’s DNA to the ribosome where protein synthesis occurs. tRNA, or transfer RNA, brings the amino acids to the ribosome’s mRNA where protein is being assembled. Lastly, rRNA, or ribosomal RNA, is the main structural element of the ribosome where the synthesis of protein occurs. As for the case of mRNA, it is classified into two kinds: the monocistronic mRNA and the polycistonic mRNA. A monocistronic mRNA, from the prefix mono-, meaning single, only one protein can be translated by the genetic information contained within it. It is a common case for eukaryotic mRNAs. On the contrary, polycistronic mRNA, from the prefix poly-, meaning numerous, many proteins can be translated by the genetic information contained in several genes. These proteins are grouped together called an operon.

In terms of structure, RNA, like DNA, is composed of an extensive chain of elements also termed as nucleotides. A nucleotide has three complex groups, namely: the nucleobase or nitrogenous base, the phosphate group, and a ribose sugar. A genetic database is solely based on the arrangement in sequence of the nucleotides. RNA has a component of a ribose sugar surrounded by 1`-5` numbered carbons. On the 1` carbon, a base is connected, namely: uracil (U), cytosine (C), adenine (A) or guanine (G). The 3` carbon of one ribose has a phosphate group attached to it while the 5` carbon is attached to the next. In which case, mRNA is just a copy of a DNA template. The mRNA typically includes the guanine cap or 5` cap, poly-adenine tail, coding region, and spliced intron and exon. On the front end of the mRNA strand, some guanine nucleotides are connected to make the ribosome bonding stronger. On the tail end of the mRNA strand, some adenine nucleotides are connected to avoid damage done by RNases (RNA breakdown of enzymes). Coding regions contain codons, proteins found in the ribosome, which are translated and decoded. It starts with a start codon and terminates with an end codon. During splicing, introns are eliminated because they are segments which have no ability to code protein while exons are combined together because they code for protein.

1.RNA is responsible in acting as a mediator in some of the biological processes of cells such as directing genetic appearance and communicating to the cell’s signals for a response. On the other hand, messenger RNA (mRNA) is a type or a particle of RNA also known as the “outline” for making protein. Messenger RNA is mainly in charge in the protein synthesis of the cell which is manufactured in the ribosome.

2.On the basis of classifications, RNA has three subtypes: mRNA, tRNA and rRNA while mRNA is classified into two kinds: the monocistronic mRNA and the polycistronic mRNA.

3.In terms of structure, RNA, like DNA, is composed of an extensive chain of elements also termed as nucleotides. A nucleotide has three complex groups, namely: the nucleobase or nitrogenous base, the phosphate group, and a ribose sugar. The mRNA typically includes the guanine cap or 5` cap, poly-adenine tail, coding region, and spliced intron and exon.


Agricultural and Related Biotechnologies

Heiner Niemann , . Björn Petersen , in Comprehensive Biotechnology (Third Edition) , 2019

4.39.4.3 RNA-Guided Genomic Engineering (CRISPR/Cas9)

The CRISPR/Cas9 system has recently emerged as potentially facile and efficient alternative to ZFNs, and TALENs for inducing targeted genetic alterations. In bacteria and archaea, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) loci encode RNA-guided adaptive immune systems that can destroy foreign DNA. 7,18 The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes, including the Cas9 nuclease and two non-coding RNAs. TracrRNA and a pre-crRNA array containing nuclease guided sequences interspaced by identical direct repeats. The design as a single transcript (single- guide RNA or guide RNA (gRNA)) encompasses the features required for both Cas9 binding and DNA target site recognition. Using sgRNA, Cas 9 can be programmed to cleave double-stranded DNA at any genomic site defined by the guide RNA sequence and a protospacer adjacent motif (PAM). The PAM is an essential targeting component that also serves as a self versus non-self recognition system to prevent the CRISPR locus itself from being targeted. Many type II systems have different PAM requirements, which may affect their usefulness and targeting efficiency. The most commonly engineered system, from Streptococcus pyogenes, requires an NGG protospacer adjacent motif (PAM), where N can be any nucleotide. In bacterial systems CRISPR/Cas can be used as it is, while in humans it involves expression of a human-codon-optimized Cas9 protein with an appropriate nuclear localization signal. Moreover, the crRNA and tracrRNA must be expressed either individually or as a single chimera via an RNA polymerase III promoter. The typical features of CRISPR/Cas9 suggest that is a simple and versatile system for generating double-stranded breaks that facilitate site-specific genome editing. Moreover, CRISPR/Cas can target multiple loci by the sgRNA, potentially allowing simultaneous targeting of multiple genomic loci. 7,18 CRISPR/Cas9 vectors are commercially available and can be used after introducing the specific gRNA sequence, which is a nucleotide of 25–30 bp.


Transcription in Eukaryotes

Fundamentally, transcription in eukaryotes is similar to transcription in prokaryotes with a few exceptions. In bacteria, RNA Polymerase can synthesize any RNA molecule. In eukaryotes, there are three different RNA Polymerases (I, II, and III). RNA Polymerase I is primarily responsible for the synthesis of ribosomal RNA ( rRNA ), the molecule that makes up ribosomes. Most eukaryotic RNA Polymerase are RNA Polymerase II. RNA Polymerase II is responsible for synthesizing mRNA, making it the only RNA Polymerase capable of transcribing protein-coding genes. RNA Polymerase III is responsible for synthesizing transfer RNA ( tRNA ). During translation, tRNAs read the messages from the mRNA and link a specific amino acid sequence generating proteins.

Where bacterial transcription is initiated by a sigma protein, RNA Polymerases in eukaryotes require a group of proteins known as basal transcription factors. Like sigma in prokaryotes, once the basal transcription factors attach to the DNA, its respective RNA Polymerase attaches and transcription begins. The elongation process is virtually identical in prokaryotes and eukaryotes. However, termination of transcription differs between prokaryotes and eukaryotes. In eukaryotes, a short sequence in the DNA signals the attachment of an enzyme downstream of active transcription. This enzyme cuts the emerging RNA, leaving the RNA Polymerase.

Post- transcriptional modification of mRNA in eukaryotes

In bacteria, transcription from DNA to mRNA is a direct pathway. However in eukaryotes once mRNA is synthesized by RNA Polymerase II, the mRNA goes through further modification (Fig. 11). The product following transcription is known as a primary transcript (or pre-mRNA ). Before mRNA travels outside the nucleus, the mRNA is shortened by cutting out specific sections of mRNA and reattaching the remaining sections back together. This process is known as RNA splicing and the resulting, modified mRNA is known as mature mRNA. Segments of the mRNA that are respliced back together are known as exons (because they exit the nucleus) while the segments of mRNA that are removed from the pre-mRNA are known as introns . The exons (which collectively make up the mature mRNA) leave the nucleus through a nuclear pore and travel to a ribosome in the cytosol and begin the process of translation.

Post-transcription modification in eukaryotes: RNA splicing

RNA splicing is processed by hybrid protein-RNA complexes known as small nuclear ribonucleoproteins (or snRNPs ). RNA splicing begins when a primary snRNP binds to a guanine R-nucleotide (G) adjacent to an uracil R-nucleotide (U) at the 5’ end of the pre-mRNA. This marks the exon-intron boundary. Another secondary snRNP reads from 5’ à 3’ down the mRNA and when it comes in contact with an adenine (A), and it attaches at that point. This point represents the intron-exon boundary. Once the primary and secondary snRNPs are attached other snRNPS attach to those, in a complex known as a spliceosome. Collectively the spliceosome breaks the G-U bond of the primary snRNP and the bond between the adenine (A) of the secondary snRNP and its adjacent R-nucleotide. Since U and A are complementary bases, the spliceosomes places them in close contact with each other, generating an intron loop. Nucleotides of the intron loop are disassembled into their monomers, R-nucleotides, and are recycled for future transcriptional events. Exons are spliced back together generating a mature mRNA.


Synthesis of RNA &ndash Transcription (With Diagram)

Genes are expressed by transfer of genetic information from DNA to RNA. From RNA the information is used for synthesizing proteins. RNA is synthesized from a portion of one strand of DNA, which acts as a template. This process is called transcription. All RNA molecules are derived from the information permanently stored in DNA except RNA genome of certain viruses.

The base sequence of RNA strand is complementary and anti-parallel to the DNA template strand. Out of the two strands of DNA only a section of one strand acts as a template and directs the synthesis of RNA. The base sequence of other strand is the same as that of RNA. The sequence of bases of RNA leads to the corresponding sequence of amino acids of protein. Thus RNA plays a central, pivotal role in the activities of the cell.

Replication and Transcription:

Basic process of chain elongation is similar in both replication and transcription. Nucleotides are added one by one in 5′ 3′ direction in both the cases. But there are some important differences.

During replication, the entire chromosome (both DNA strands) is copied but the transcription is more selective. One particular gene or group of genes is transcribed at any one time, producing one to numerous number of copies. Some parts of DNA are never transcribed. In transcription, RNA strand is made up of ribonucleotides rather than deoxyribonucleotides. Transcription does not require a primer but, replication needs primer for copying both strands.

Process of RNA Synthesis:

The basic features of RNA synthesis are as follows:

1. The building blocks of RNA are four ribonucleotide 5-triphosphates. They are ATP, GTP, CTP and UTP.

In polymerization, ribonucleotides are added one-by-one. The 3′-OH group of one nucleotide reacts with 5′-triphosphate of next nucleotide, the pyrophosphate (PP) is released. This is similar to DNA polymerization in DNA replication.

2. The sequence of bases in RNA is determined by the base sequence of DNA template strand. The base sequence of RNA strand is complementary to the template strand of DNA. Thymine is replaced by Uracil in RNA.

3. Out of two strands of DNA, often one strand acts as a template for RNA synthesis. Transcription selectively copies only certain parts of genome and makes one to numerous copies of RNA. The RNA formed is antiparallel to the DNA template.

4. Unlike DNA replication, which needs RNA primer, RNA polymerase enzyme, which synthesizes RNA strand does not need a primer and can initiate transcription de novo.

RNA synthesis consists of four stages:

1. Binding of RNA polymerase enzyme to the template at a promoter site on DNA.

2. Initiation of new strand.

4. Termination and release.

Transcription in Prokaryotes:

Length of mRNA chain depends upon the length of polypeptide chain it codes for. The mRNA in prokaryotes is generally polycistronic which codes for several proteins which represent proteins of a single metabolic pathway.

RNA Polymerase Enzymes:

DNA dependent RNA polymerase enzyme requires a DNA template. In addition, it requires four ribonucleotide-triphosphates which are ATP, GTP, UTP and CTP. Each nucleotide in the newly formed RNA strand is selected on the basis of Watson-Crick base pairing rule.

In prokaryotes all types of RNAs are transcribed by the same RNA polymerase enzyme RNA polymerase is one of the largest enzymes having a molecular weight 500000 daltons. When it binds DNA, it covers many bases of DNA simultaneously.

RNA polymerase consists of subunits-2α subunits, β, β and c (sigma factor). Complete enzyme is called holoenzyme. The sigma factor separates from the holoenzyme. The enzyme without factor sigma factor is called core enzyme.

There is a definite sequence of bases on DNA called promoter to which the RNA polymerase binds. RNA polymerase recognise this promoter and then locally unwinds the DNA strands in order to gain access to the bases to be copied. In prokaryotes, the promoter is a sequence of six bases which in generally TATAAT or its slight variant.

This sequence is called Pribnow box. It lies 5-10 bases before the first base to be transcribed and is also called – 10 region. This region is called upstream region and is denoted by a minus sign (-).

Pribnow box orients RNA polymerase in such a way that transcription proceeds in 5′ → 3′ direction. In addition several promoters have a second important region to the left of Pribnow box which is a – 35 sequence. It is another six base sequence TTGACA. The large RNA polymerase enzyme covers – 35 sequence, Pribnow box and transcriptional start site.

These two conserved sequences called – 35 and – 10 regions are 17-19 nucleotides apart.

After initiation, elongation of RNA chain occurs. New ribonucleotides are added one by one. It is called polymerization. During elongation RNA polymerase copies DNA sequence accurately. This is known as processivity. Elongation of RNA proceeds only in 5′ —> 3′ direction like DNA replication.

During elongation phase of transcription, the RNA strand base pairs temporarily with DNA template to form a short RNA-DNA hybrid double helix. Then the RNA separates and two strands of DNA again form duplex structure. DNA strands must unwind over a short distance to enable RNA polymerase to synthesize RNA strand complementary to one of DNA strands.

This unwinding of DNA helix opens up the DNA helix. A wave of unwinding is generated by RNA polymerase. In this way DNA is unwound ahead and rewound behind as RNA is transcribed.

RNA polymerase also performs proof reading functions as transcription is less accurate than replication. In replication one mistake is made for every 10,000,000 nucleotides added while in transcription one mistake is made for every 10000 nucleotides added.

Termination and Release:

After the RNA synthesis is completed, RNA polymerase reaches a stop signal or a termination factor on DNA. In E. coli there are two types of termination signals, Rho-independent and Rho-dependent.

In Rho-independent termination is also called intrinsic termination. Near the 3′-end of newly synthesized RNA, there is a sequence of inverted repeats, about 20 nucleotides long, which form a self complementary hairpin or stem structure. At the end of this hairpin structure there is a sequence of uracil bases which are transcribed from adenine bases on the template strand. This A = U hybrid region has weakest bonds, and therefore is unstable and leads to the termination and dissociation of newly synthesized RNA molecule. This is self-terminating and depends upon the DNA base sequence.

Rho-dependent termination is dependent on a protein factor called Rho factor. Here Rho- protein has an ATP-dependent RNA-DNA helicase activity that causes release of newly synthesized RNA molecule.

In Prokaryotes Transcription and Translation Go On Simultaneously:

As prokaryotes do not have a nuclear membrane, transcription and translation occur in the same compartment. Even as the synthesis of RNA is in progress, ribosomes bind to the free 5′-end of mRNA and start protein synthesis or translation. The ribosomes bind at the free 5′-end mRNA while the 3′-end is still being transcribed. This is called coupled transcription and translation. Transcription, translation and degradation of mRNA all occur in the same 5′ → 3′ direction.

Transcription in Eukaryotes:

The basic features of transcription and structure of mRNA in eukaryotes are similar to those of bacteria. But there are certain differences between prokaryotes and eukaryotes. In eukaryotes, the mRNAs are generally monocistronic as compared to polycistronic mRNA of prokaryotes.

RNA Polymerase of Eukaryotes:

There are three kinds of RNA polymeases in eukaryotes. They are RNA polymerase 1, RNA polymerase II and RNA polymerase III.

RNA polymerase I transcribes only rRNAs i.e., 5.8S, 18S, and 28S, rRNAs. The RNA polymerase II transcribes all mRNAs. The RNA polymerase III transcribes tRNA and 5S rRNA.

Promoters for RNA Polymerase II:

The promoters for RNA polymerase II in eukaryotes are more complex. One promoter consists of a sequence of bases which lie – 25 bases upstream of transcription starting site. It consists of a sequence of seven bases TATAAAT called TATA box. It is also called Hogness box. It can be compared to Pribnow box of prokaryotes. Only TATA box is present in almost all the eukaryotes. In many cases another sequence is also present which lies – 75 base region called CAAT box. It has a sequence GGTCAATCT. Transcription start site lies in the initiator sequence where DNA is unwound.

Various transcription factors bind to TATA box and other sequences. RNA polymerase does not bind to these promoter sequences directly but to these factors as eukaryotic DNA is in the form of chromatin.

Post Synthesis Processing of RNA:

Newly synthesized RNA is called primary transcript or precursor RNA (pre-RNA). These RNA molecules undergo extensive changes called or processing to form mature RNA molecules which take part in protein synthesis. The most complex and characteristic feature of pre-mRNA is the presence of non-coding regions called introns present in between coding regions called exons.

These non-coding regions or introns are removed and discarded before the protein synthesis can take place. They are removed by a process called splicing. This is essential to get a correct sequence of amino acids of a polypeptide.

Almost all kinds of RNA molecules undergo post synthesis processing. Prokaryotic mRNA is generally not processed. Eukaryotic mRNA undergoes maximum processing. Both prokaryotic and eukaryotic tRNAs and rRNAs undergo extensive processing.

Roger Korenberg of America won Nobel Prize for Chemistry for describing the molecular basis of transcription in eukaryotic cells, how the information in the genes is copied and transferred.

His father Arthur Korenberg had won Nobel Prize in medicine in 1959 also for genetic work.

Summary:

Genes are expressed by transfer of genetic information from DNA to RNA. From RNA the information is used for synthesizing proteins. RNA is synthesized from a portion of one
strand of DNA which acts as a template. One gene or group of genes is transcribed at any one time producing one to numerous copies. On the other hand in replication both DNA strands are copied to produce two copies similar to the parent DNA molecule.

Polymerization is similar in both replication and transcription. Ribonucleotides of RNA are ATP, GTP, CTP, UTP. Thymine of DNA is replaced by uracil in RNA. RNA strand formed is complementary and antiparallel to the DNA template strand. Unlike replication, no primer is required in transcription. In prokaryotes a single RNA polymerase enzyme synthesizes all types of RNAs.

There is a definite sequence of bases on DNA called promoter on which the RNA polymerase binds. In prokaryotes a sequence of six bases called Pribnow box lies 5-10 bases before the transcriptional start site. Another sequence called – 35 sequence may be present. RNA polymerase binds these promoter sites and unwinds the DNA molecule for copying the portion of DNA template.

In E. coli two types of termination signals, Rho-independent and Rho- dependent signals are present. In prokaryotes, transcription and translation go on simultaneously. In eukaryotes basic steps of transcription are similar to prokaryotes. But some major differences are there. In eukaryotes three kinds of RNA polymerase enzymes are present. RNA polymerase I transcribes only rRNAs i.e. 5.8S, 18S and 28S rRNAs. The RNA polymerase II transcribes all mRNAs.

The RNA polymerase III transcribes tRNA and 5S rRNA. Promoters for RNA polymerase II are -25 sequence called TATA box and – 75 sequence called CAAT box. Newly synthesized RNA is called primary transcript or pre-RNA. These RNA molecules undergo extensive changes called processing to form mature RNA molecules which take part in protein synthesis. Processing occurs by splicing in which non coding regions called introns are spliced out and coding regions or exons are joined.


Transcription: DNA → RNA

Transcription is the first half of the Central Dogma. This is where DNA is translated into RNA. Transcription occurs in the nucleus of the cell—DNA cannot leave the nucleus. There are three steps in transcription: initiation, elongation, and termination (these are also the same steps as in translation however, different things happen in the steps of the different processes).

  1. Initiation: Transcription begins at a promoter: a specific region of a gene. RNA polymerase binds to the promoter. This signals the DNA to unwind. The enzyme is now ready to make mRNA
  2. Elongation: Nucleotides are added to the mRNA strand
    • Remember: thymine only occurs in DNA, and uracil only occurs in RNA!
  3. Termination: Transcription ends when RNA polymerase encounters a stop (termination) sequence in the gene.

RNA Polymerase

There are three types of eukaryotic RNA Polymerase. Fittingly, they’re named RNA Polymerase I, RNA Polymerase II, and RNA Polymerase III.

  • RNA polymerase I is located in the nucleolus, and facilitates the transcription of ribosomal RNA (rRNA), which is then processed and assembled into ribosomes.
  • RNA polymerase II is located in the nucleus and synthesizes all protein-coding nuclear pre-mRNAs.
  • RNA polymerase III is also located in the nucleus. This polymerase transcribes a variety of structural RNAs that includes the 5S pre-rRNA, transfer pre-RNAs (pre-tRNAs), and small nuclear pre-RNAs.

MRNA Processing

After transcription, eukaryotic pre-mRNAs must undergo several processing steps before they can be translated.

Pre-mRNAs are first coated in RNA-stabilizing proteins these protect the pre-mRNA from degradation while it is processed and exported out of the nucleus. The three most important steps of pre-mRNA processing are the addition of stabilizing and signaling factors at the 5′ and 3′ ends of the molecule, and the removal of intervening sequences that do not specify the appropriate amino acids. In rare cases, the mRNA transcript can be “edited” after it is transcribed.

PRactice Questions

In which step of transcription does the DNA unwind?

Which is a function of RNA polymerase II?

  1. transcribes transfer pre-RNAs (pre-tRNAs)
  2. facilitates the transcription of ribosomal RNA (rRNA)
  3. synthesizes all protein-coding nuclear pre-mRNAs

Affiliations

Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14 Str., 61-704, Poznan, Poland

Edyta Koscianska, Julia Starega-Roslan, Lukasz J Sznajder, Marta Olejniczak, Paulina Galka-Marciniak & Wlodzimierz J Krzyzosiak

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Corresponding author


Watch the video: mRNA Splicing (May 2022).


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