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Molecular Science. Chapter 3 Pages 69-83 Mikhail Kotlik. Review of Chapter 2 & Unanswered questions. The previous chapter covered: The basic mechanisms of DNA replication, transcription, and translation. Science still had not revealed:
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Molecular Science Chapter 3Pages 69-83 Mikhail Kotlik
Review of Chapter 2 & Unanswered questions The previous chapter covered: • The basic mechanisms of DNA replication, transcription, and translation. Science still had not revealed: - How these mechanisms were regulated - Why is a specific gene transcribed at a specific moment or in a specific cell?
Jacob, Monod, & the Lac Operon • In the late 1950s and the early 1960s, Parisian researchers François Jacob and Jacques Monod began the study of gene regulation by researching how lactose metabolism genes are regulated. • Provided the E. coli bacteria, which normally consumes glucose, with lactose instead. • As a result, the bacteria quickly began producing three proteins which are normally not present in the organisms, and are necessary for the breakdown of lactose into glucose and galactose.
Jacob, Monod, & the Lac Operon • The genes for the three proteins, lie in tandem on the bacterial chromosome and are transcribed onto a single mRNA. - An mRNA molecule coding for multiple protein is called a polycistronic mRNA. • A cluster of genes is called an operon. - In the case of lactose metabolism, the genes for the proteins that metabolize lactose are collectively known as the lac operon.
Jacob, Monod, & the Lac Operon • Jacob and Monod performed a series of experiments to determine how these genes are switched on in the presence of lactose. • In one experiment, the researchers created bacteria containing partial diploids (2 copies) of the lac genes. - E. coli are normally haploid bacteria. In addition to a regular chromosome, they can contain smaller plasmids. The F plasmid (Fertility plasmid) is similar to a sex chromosome, and codes for a pilus. A pilus, is a structure that allows two bacterial cells to share genetic information. After the pilus is created, the F plasmid can bind to either an entire chromosome or specific genes, and carry them to recipient cell. The recipient cell can become either fully diploid, or partially diploid for certain genes.
Jacob, Monod, & the Lac Operon • The researchers created several partial diploid strains of E. coli with an F’ plasmid (a genetically engineered plasmid) and genomic DNA that contained wild-type (lac+) or mutant (lac-) versions of the genes in the lac operon. • They grouped the varying strains of modified E. coli based on the ability of their lac genes to be regulated by lactose and metabolize it. • One class of genomic mutant strains, were those who could metabolize lactose if a wild-type version of a lac gene was present on the F’ plasmid. - This demonstrated that such lacgene mutations were recessive.
Jacob, Monod, & the Lac Operon • Another class of genomic mutant strains, were those who expressed lac genes regardless of the presence of lactose (constitutive expression), and were grouped into two types. - One type of mutations was recessive, such that the presence of a wild-type gene on the F’ plasmid restored lactose-dependent expression. Called trans mutations (assert their dominance across a distance/across DNA molecules). Mutations were located within the lac repressor (lacI) gene, coding for the lac repressor protein that binds to DNA and blocks the expression of genes coding for lactose metabolism proteins. - The second type of mutations was “dominant-like”. The mutations acted “in cis”, as they affected only the genes located on the same DNA as the mutation. The mutations were located in the lacO(operator) region of the DNA, which is a region at the beginning of one of the lac genes and to which the Lac repressor protein binds. This type of mutation prevented the Lac repressor from binding, and allowed for constitutive expression of the lac genes.
Jacob, Monod, & the Lac Operon • The final class of mutations also acted in cis. E. coli strains with these mutations could not express lac operon genes. Located in lacP (promoter) region, which is outside of the lacO region. The promoter region is recognized by RNA polymerase as the site at which to begin transcription.
Jacob, Monod, & the Lac Operon Lac Operon Structure • The first region of the operon is the CAP site, to which the CAP protein (a regulatory protein) binds. In the absence of glucose, CAP stimulates expression of the lac genes by assisting polymerase in binding to the promoter, and increasing transcription (positive regulation of gene expression).- The second region is the lac repressor gene. In the absence of lactose, the lac repressor protein (a regulatory protein), binds to the operator region and prevents RNA polymerase from moving further down the DNA (negative regulation). When lactose is present, it binds to the lac repressor and causes a structural change, releasing it from the operator, and allowing transcription. • These regions are followed by the three lac genes.
Jacob, Monod, & the Lac Operon • Negative gene regulation introduced the concept of a promoter. - Promoters can be found in all genes. - However negative regulation is uncommon, and nonexistent in eukaryotes.
Eukaryotic Gene Regulation • Gene regulation is more complicated in eukaryotes than in bacteria- RNA polymerase can only bind to DNA along with a group of other proteins- Regulatory proteins bind not only at the promoter region but also throughout the DNA, at enhancer sites.- Eukaryotic DNA is intertwined with several types of protein, in a structure called chromatin. This structure can prevent regulatory proteins and transcription “machinery” from getting access to the DNA. - Chromatin can be chemically modified by enzymes found in the cell to allow the binding of polymerase and other factors. • Eukaryotes have 3 different RNA polymerases, each with a different role. - Pol I transcribes the large ribosomal precursor RNA - Pol II transcribes primarily mRNA and a few specialized RNA molecules. - Pol III transcribes tRNAs, and other small RNAs.
Eukaryotic Gene Regulation Initiation of Transcription- The promoter region of a eukaryotic gene consists of 3 distinct parts.- The first part is the TATA box, consisting of a repeating “TATAAAA” sequence (with minor variations). Multiple factors associated with Pol II bind to the TATA box. - TATA-binding protein (TBP) is a protein that is part of the TFIID complex, and binds to the TATA box. TBP is essential for the initiation of transcription and forms a central part of the preinitiation complex. After binding to the TATA box, it bends the DNA and forces the helix to open slightly, giving Pol II better access.
Eukaryotic Gene Regulation Transcriptional Regulation by Transcription Factors- Transcription factors are regulatory proteins- Some bind close to the promoter, whereas others bind to more distant enhancer elements.- Regulation of a specific gene requires the binding of multiple factors. Ex. The GC box is a common element in eukaryotic promoters, and is bound by the activator Sp1. Ex. The CCAAT box is also a common element, and is bound by the activator C/EBP.- Transcription factors that promote transcription are called activators. - Activators bind to DNA with one surface and interact with factors associated with Pol II with another. Thus, they help Pol II bind to the promoter and initiate transcription.- Transcription factors that reduce transcription are called repressors.- Transcription factors probably control the rate at which Pol II releases from the initiation complex to begin transcription.- Transcription factors are controlled by signals (ex. growth factors) in a variety of ways, including entry of the factors into the nucleus and their activity after they are bound to DNA.
Eukaryotic Gene Regulation Enhancers- Enhancers also bind activators and repressors, at a distance from the gene.- Can be moved or flipped without affecting their function.- Activators bound to enhancers interact with Pol II or other initiation factors at the promoter, and activate in a manner similar to activators bound closer to the promoter. - To allow activation over a great distance, the DNA between the enhancer and the promoter forms a loop.
Eukaryotic Gene Regulation Signal Integration at Enhancers- Activators bound to an enhancer can work together to switch on a gene (signal integration).- Ex. The enhancer that controls the human interferon-ß gene (its product helps fight viral infection). - When a virus enters a cell, it triggers 3 different activators that bind adjacent to each other at an enhancer. Each activator helps the others bind, and they only bind if all 3 are present (cooperative binding).- The activators enhance each others’ ability to bind DNA through interaction. An additional protein binds to the enhancer, bending the DNA in a way that allows the activators to bind and interact.
RNA Processing - As an mRNA is being synthesized, it is being further processed with the addition of a 5’ cap structure. After transcription, it is further processed with the addition of a poly(A) tail. - The processing partially determines the life span of the mRNA.Creating a 5’ Cap- The cap stabilizes the mRNA, and is involved in mRNA splicing, export of mRNA from the nucleus, and recognition of mRNA by translation machinery during protein synthesis.- The cap is composed of a guanosine (G) nucleotide covalently attached to the 5’ phosphate of the 1st nucleotide of the DNA. The guanine base is then methylated at position N7. The creation process involves 3 separate enzymatic reactions. - Firstly, RNA 5’-triphosphate removes 2 of the 3 phosphates that are contained on the 5’ end of the mRNA after synthesis, creating a monophosphate. - Secondly, mRNA guanylyltransferase (capping enzyme) ligates a guanosine diphosphate (GDP) to the 5’ monophosphate. - Thirdly, RNA guanine-7-methyltransferase methylates N7 of the guanine base.
RNA Processing Creating a Poly(A) Tail- The 3’ end of almost all mRNA molecules includes a long stretch of adenosines.- The Poly(A) tail stabilizes the mRNA. It is often used by scientists in isolating and working with mRNA.- The enzyme polyadenylate polymerase recognizes the sequence AAUAAA. The enzyme then cleaves the mRNA 11-30 bases in the 3’ direction from this sequence. Lastly, the enzyme adds a long stretch of adenosines to the 3’ end.
RNA Processing mRNA Stability and Turnover- mRNA is a short-lived messenger that is rapidly degraded (“turned over”).- The number of proteins produced by a single mRNA is partially dependent on the length of time the mRNA exists before being degraded.- The mRNA half-life is the time needed for half of a population of mRNAs to degrade. - Ex. mRNA coding for a- and ß-globin have very long half-lives of hours to days. mRNAs of some regulatory proteins have half-lives that last seconds.- The Poly(A) tail is one factor that determines mRNA half-life. It gradually degrades as an mRNA “ages”, and the entire molecule degrades when the tail shortens to a certain length.- The 3’-noncoding region of DNA can also be involved in eukaryotic mRNA stability. - A sequence rich in adenine and uracil (AU) residues is frequently found in that region of transiently expressed genes. The sequence is a signal for selective degradation of mRNA by 3’ nuclease from the mRNA’s 3’ end.
RNA Processing mRNA Stability and Turnover- Altering regulation of mRNA half-life can have devastating developmental effects on a cell.- One experiment studied the fos oncogene, which is mutated in some cancers. - The mRNA of the normal cellular gene (c-fos), has a very short half-life and has an AU-rich sequence. - The mRNA of a retrovirus version of the gene (v-fos), has a much longer half-life and no AU-rich sequence. - The v-fos gene and it’s similar human mutant may cause cancer by prolonging the presence of the fos protein in a cell.- The c-fos mRNA has also been shown to have a region called mCRD, which can also cause degradation of the mRNA through interaction with the Poly(A) tail.
RNA Processing RNA Splicing- In 1976, Jerry Lingrel, Jeffrey Ross, and Charles Weismann, discovered a ß-globin pre-cursor RNA in the nucleus that was larger than ß-globin mRNA. The precursor was then processed into smaller, mature mRNA. This supported earlier findings that the nucleus contains large RNA molecules, called heterogeneous nuclear RNA (hnRNA).- At the same time, two groups, one from the Cold Spring Harbor Laboratory and one from MIT, found that genes and pre-mRNAs contain 1 or more blocks of sequence, called introns, that are not found in mature mRNA.- Through RNA splicing, introns are edited out and the remaining pieces of sequence, called exons, are joined together to form mRNA.- This occurs in the genomes of most eukaryotes.
RNA Processing RNA Splicing- Other investigators showed that introns are excised in a 2-step process of sequential trans-esterification reactions. At the end of these reactions, two exons are joined together and the intervening intron is released as a branched lariat structure.- The cellular splicing machinery is made up of five different U-rich small nuclear RNAs (snRNAs), and more than 60 proteins. - Several observations showed that U-rich snRNAs assist in the splicing reaction. Firstly, a sequence at the 5’ end of introns was found to be complementary to a sequence near the 5’ end of the U1 snRNA. Secondly, snRNAs were found to be associated with hnRNAs.
