Biotechnology Unit 3: DNA to Proteins Essential Cell Biology ...

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Essential Cell Biology Chapter 7. From DNA to RNA. I. After the discovery of the structure of DNA, the major question remaining was how does the information ...
Biotechnology Unit 3: DNA to Proteins From DNA to RNA I.

After the discovery of the structure of DNA, the major question remaining was how does the information stored in the 4 letter code of DNA direct the development and function of an entire organism II. The central dogma of molecular biology a. There are 2 general steps to the production of a protein from a gene i. Genes code for RNA (transcription) ii. RNA codes for the polypeptide (translation) b. So, the flow of genetic information is from DNA  RNA  Protein i. This is known as the central dogma of molecular biology c. Together, transcription and translation are the means by which cells express their genetic information i. This is known as gene expression III. Transcription of DNA into RNA a. RNA is an intermediate produced from DNA in the process of protein synthesis i. RNA can have many forms and an incredibly wide range of functions within the cell b. Like DNA, RNA is a linear polymer made up of four different nucleotides linked together by phosphodiester bonds i. There are two major chemical differences between DNA and RNA 1. The nucleotides in RNA are ribonucleotides instead of deoxyribonucleotides a. They contain the 5-carbon sugar ribose instead of deoxyribose 2. The bases involved in the ribonucleotides are A, C, G, and U (uracil) a. Uracil replaces thymine and complimentarily binds with adenine with 2 hydrogen bonds just like thymine ii. There is also a fairly major structural difference between DNA and RNA 1. RNA is single stranded instead of being a double helix like DNA a. This allows RNA to fold into many different shapes to accomplish many different functions c. All RNA in a cell is made by transcription i. There are several similarities between transcription and DNA replication 1. A small portion of the DNA double helix is opened up and unwound to expose the bases of the DNA 2. One of the two strands acts as a template and ribonucleotides are added using complimentary base-pairing 3. The new RNA molecule (called a transcript) grows one nucleotide at a time a. The enzyme responsible for reading the template and building the new RNA molecule is called RNA polymerase and it reads from 3’ to 5’ and builds from 5’ to 3’ like DNA polymerase i. RNA polymerase uses energy from ribonucleoside triphosphates for energy just like DNA polymerase ii. Transcription does differ from DNA replication in some crucial ways 1. The RNA transcript that is formed does not stay hydrogen bonded to the template strand like DNA does a. The RNA ends up being single stranded b. Just behind the region where nucleotides are being added, the RNA transcript is released and the DNA double helix reforms Essential Cell Biology Chapter 7

Biotechnology Unit 3: DNA to Proteins 2. Only one side of the DNA molecule acts a template during transcription a. Both sides can be templates for different genes at different locations on the chromosome however b. The template or antisense strand is the strand that is read by the RNA polymerase c. The other strand is called the coding or sense strand and is the model for the RNA molecule 3. RNA molecules are only transcribed from limited regions of DNA a. This means that transcription only produces a complimentary segment of not more than a few thousand base-pairs and not an entire DNA molecule 4. Transcription of the same gene can occur simultaneously by several different RNA polymerases a. Because the transcripts are released almost immediately, transcription can start over shortly after the RNA polymerase has moved on from the starting point b. It takes approximately 50 seconds to transcribe a molecule of RNA from a medium sized gene of 1500 nucleotide pairs i. As many as 15 RNA polymerases can simultaneously be transcribing this gene at any one give time ii. This means over 1000 transcripts can be synthesized in a hour 5. RNA polymerase is very similar in its action as DNA polymerase but there are a couple key differences a. RNA polymerase polymerizes ribonucleotides instead of deoxyribonucleotides b. RNA polymerase does not require RNA primers i. Because RNA does not permanently store genetic information, the necessity of accuracy in transcription is lower than in DNA 1. RNA polymerases make approximately 1 mistake in every 104 nucleotides compared to the rate of one mistake in 107 nucleotides by DNA polymerase iii. Several types of RNA are produced in cells 1. The vast majority of genes in a cell’s DNA code for the amino acid sequence of proteins a. The RNA molecules that are copied from these genes are called messenger RNA (mRNA) i. In eucaryotes, each mRNA carries information from just one gene, coding for just one protein ii. In procaryotes, several adjacent genes are often transcribed together into a single mRNA which carries the information for several different proteins 2. In other genes, the final product of the gene is the RNA itself a. These nonmessenger RNAs can serve a regulatory, structural, and enzymatic components of cells and they are important to the translation of proteins and the regulation of gene expression b. Ribosomal RNA (rRNA) forms the central core of the ribosomes where mRNA is translated into proteins c. Transfer RNA (tRNA) for the adaptors that hold the amino acids in place on the ribosome so they can be incorporated into the protein d. MicroRNAs (miRNAs) serve as regulators of eukaryotic gene expression d. Signals in DNA tell RNA polymerase where to start and finish Essential Cell Biology Chapter 7

Biotechnology Unit 3: DNA to Proteins i. Because of the fact that not all of the DNA of an organism codes for proteins and because not all of the genes are active in all cells, there has to be a mechanism to tell RNA polymerase where to begin transcription ii. The beginning of a gene must be signaled in order for transcription to begin iii. This process is different in procaryotes and eucaryotes 1. In procaryotes, when RNA polymerase collides with a DNA molecule it attaches itself very weakly and moves along the DNA until it finds a specific region called a promoter a. At the promoter, the RNA polymerase binds tightly and opens up the double helix in front of itself i. Promoter regions are asymmetrical which prevents transcription from occurring in any direction other than 5’ to 3’ b. The RNA polymerase then moves along the DNA double helix reading the template strand and producing the complimentary RNA transcript until it reaches another specific region called the terminator or stop site i. At the terminator site, the RNA polymerase releases from the DNA double helix c. The procaryotic RNA polymerase has a special subunit called a sigma factor that helps to identify the promoter region i. Once the promoter is identified, the sigma factor disengages itself from the RNA polymerase until transcription is finished and the polymerase has disengaged 2. In eucaryotes, the process is much more complex a. Unlike procaryotes, eucaryotes have three different RNA polymerases i. RNA polymerase I and RNA polymerase III are responsible for transcribing tRNA, rRNA, and miRNAs ii. RNA polymerase II is responsible for transcribing mRNA b. Another difference from procaryotes is that eukaryotic RNA polymerase doesn’t have a sigma factor and therefor is dependent upon a large group of accessory proteins called transcription factors which assemble at the promoter before transcription can occur i. A group of general transcription factors assemble at a short section of DNA located 25 base-pairs upstream of the gene 1. This region is usually rich in A and T nucleotides and is called the TATA box ii. Once the RNA polymerase II is bound to the transcription factor complex, it is phosphorylated by adding a phosphate to its tail and that allows it to break away from the transcription factors and begin transcription 1. The transcription factors then release and are available to reform at another promoter region c. The geography of eukaryotic chromosomes also affects transcription because the individual genes are spread out considerably more than they are in procaryotes i. This allows for almost unlimited combinations of regulatory sequences for individual genes d. Finally, the packing of the eukaryotic chromosomes adds a level of complexity to transcription e. Processing of transcripts i. In prokaryotes, the DNA is already in the cytoplasm and exposed to ribosomes Essential Cell Biology Chapter 7

Biotechnology Unit 3: DNA to Proteins 1. Ribosomes can attach to the 5’ end of the transcript and begin the process of protein synthesis while transcription is still going ii. In eucaryotes, the DNA is enclosed inside the nucleus and is not exposed to the ribosomes until it leaves 1. Eucaryotic transcripts go through several steps of RNA processing before they can leave the nucleus a. These steps take place while transcription is occurring because the enzymes are attached to the tail of the RNA polymerase b. RNA capping involves adding methylated guanines to the 5’ end of the transcript c. Polyadenylation involves cutting a small portion off of the 3’ end of the transcript and then adds a few hundred adenines creating a poly-A tail d. Both RNA capping and polyadenylation are thought to add stability and to help with the transport of the mRNA out of the nucleus

2. Most eukaryotic genes are interrupted with noncoding regions that need to be removed from the transcript before translation can occur a. The noncoding regions are called introns and the coding regions are called exons i. The introns are usually much longer than the exons b. The entire length of a gene is transcribed including both introns and exons c. The introns need to be removed by RNA splicing i. There are sequences within the introns that identify them as the regions that need to be cut out ii. The splicing of mRNA is done by other RNA molecules called small nuclear RNAs (snRNAs) that combine with proteins to form small nuclear ribonucleoprotein particles (snRNPs) (pronounced “snurps”) iii. The cutting follows what is known as a lariat cut 3. Alternative splicing provides the ability for one mRNA to be used to produce several different proteins Essential Cell Biology Chapter 7

Biotechnology Unit 3: DNA to Proteins a. Splicing the RNA differently (keeping different exons) provides many different functional mRNA molecules b. It is estimated that 60% of human genes undergo alternative splicing

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iii. Once RNA processing and splicing is complete, the mRNA must leave the nucleus to join with ribosomes to be translated into proteins Translation is the process of converting the RNA sequence into a protein a. Translation involves a much more dramatic change than transcription i. The “alphabet” used to carry the code in DNA and RNA is based on 5 (but only 4 at a time) nucleotides ii. The “alphabet” used to build proteins is composed of 20 amino acids 1. There cannot be a one to one relationship between RNA and proteins so we need what is referred to as the genetic code that is identical in nearly all life forms a. The mRNA sequence is read in consecutive groups of 3 nucleotides called codons i. There are 4x4x4 = 64 possibilities for codon combinations ii. Because there are only 20 amino acids, there is a built in redundancy meaning come amino acids are coded for by more than one codon b. This allows for three unique reading frames depending on where the reading begins i. There is a specific start codon that identifies the proper reading frame for each protein b. The rapid translation of mRNA into a protein requires a molecular machine that moves along the RNA, captures and holds tRNA molecules, and covalently links the incoming amino acids i. Transfer RNA (tRNAs) molecules match amino acids to the proper mRNA codons 1. tRNAs are molecules roughly 80 nucleotides long that act as adapters to bring in the correct amino acid that is coded for by each codon a. Each tRNA contains a region known as the anticodon which is complimentary to the codon on the mRNA b. The 3’ end of the tRNA holds a specific amino acid molecule c. tRNA molecules are said to be charged when the amino acid is attached i. This is done by enzymes called aminoacyl-tRNA synthetases Essential Cell Biology Chapter 7

Biotechnology Unit 3: DNA to Proteins ii. Each amino acid has a specific synthetase that is responsible for connecting that amino acid to the tRNA molecules with the proper anticodons ii. Ribosomes are the protein manufacturing machine 1. They are made of over 50 different proteins and several ribosomal RNA (rRNA) molecules 2. Typical cells have millions of individual ribosomes a. Both eucaryotic and procaryotic ribosomes are similar and are made up of a large subunit and a small subunit i. The small subunit pairs the tRNAs to the mRNA codons ii. The large subunit links the amino acids into the polypeptide chain b. The ribosome attaches near the 5’ end of the mRNA and then pulls the mRNA through while it attaches the appropriate amino acids i. Eucaryotic ribosomes can add about 2 amino acids per second ii. Procaryotic ribosomes can combine up to 20 amino acids per second 3. Along with the binding site for the mRNA, the ribosome has three sites for tRNAs a. The A-site is where the new tRNA comes in and binds to the anticodon and brings in the new amino acid b. The P-site is where the previous tRNA is held until the new amino acid can be linked onto the chain c. The E-site holds the tRNA until it is ejected d. The mRNA moves through these sites repeatedly until the polypeptide chain is complete 4. Codons in mRNA signal where to start and stop protein synthesis a. Translation always begins with the codon AUG i. The initiator tRNA carries the amino acid methionine and attaches to the AUG codon ii. This initiator tRNA is different from the normal methionine carrying tRNA iii. The small ribosomal subunit attaches to the 5’end of them RNA (signaled by the 5’ cap) and then reads along until it finds an AUG b. It then reads along adding all the necessary amino acids until it reaches a stop codon i. UAA, UGA, and UAG are the stop codons ii. Stop codons do not correspond to a tRNA and instead signal the ribosome to stop translation iii. Release factors (proteins) bind to the stop codon in the A-site and cause the polypeptide and the ribosome to be released from the mRNA

Essential Cell Biology Chapter 7

Biotechnology Unit 3: DNA to Proteins

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Essential Cell Biology Chapter 7

Biotechnology Unit 3: DNA to Proteins Overview of Gene Expression I.

Every cell in an organism has a complete set of the genes for that organism but not all of the proteins are needed within the cell a. Different cell types produce different sets of proteins b. The amount of each protein that the cell needs is different at different times c. The control of gene expression is how the cell deals with these issues i. Cells can change the expression of genes in response to external signals II. Gene expression can be regulated at many different levels or step within the pathway from DNA to RNA to protein a. Transcriptional control i. Transcription is controlled by proteins binding to regulatory DNA sequences 1. Remember that in order for transcription to begin, RNA polymerase must attach and orient itself at the promoter region of the gene 2. The simplest regulatory sequences are only 10 nucleotides long a. These are most common in bacteria 3. In eucaryotes, the regulatory sequences can be up to 10,000 nucleotide pairs and can interact with a wide variety of signals 4. Transcription regulators are proteins that bind to specific regulatory DNA sequences to act as a switch for the control of transcription a. Different proteins recognize different sequences based on the shape of the protein ii. In procaryotes, operons control the expression of groups of genes 1. There is a region at the start of the operon called the operator which is where transcription of the entire group of genes will begin 2. Operators are controlled by repressors and activators a. A repressor is a protein that when bound to the operator does not allow transcription to begin by blocking the attachment of RNA polymerase b. An activator is a protein that allows transcription to occur by allowing for better attachment of the DNA polymerase c. Some operons use both repressors and activators 3. Operon examples a. Tryptophan operon i. This operon uses a repressor and it controls a group of five genes that are necessary for the production of the amino acid tryptophan ii. When tryptophan is in abundance in the cell, it can bind to the repressor protein which will then bind to the operon and prevent transcription iii. When tryptophan is in low concentration, it cannot bind to the repressor protein and the repressor protein then cannot bind to the operator and transcription will occur allowing for the production of tryptophan

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Biotechnology Unit 3: DNA to Proteins

b. Lac operon i. This operon uses both a repressor and an activator to control the transcription of several genes used to break down lactose which is a food source for bacteria ii. Glucose is the bacterial cells first choice for food so when glucose is presence the Lac operon is switched off because it is lacking a necessary activator protein that is only available when glucose is absent iii. The operon is also switched off by a repressor protein that can only be removed when lactose is present iv. The only time the Lac operon is active, is when both the activator is bound (glucose not present) and the repressor is not bound (lactose is present)

Essential Cell Biology Chapter 8

Biotechnology Unit 3: DNA to Proteins iii. Eucaryotes also use transcription regulators (both repressors and activators) but they do not use operons to control groups of genes 1. These sites are called enhancers 2. Eucaryotic transcription regulators can work from a distance a. The binding of activators or repressors in eucaryotes does not need to be close to the gene of interest or even be upstream from it b. In many instances the transcription regulators are long distances away but there is a looping that occurs to allow for the initiation of transcription b. Post-transcriptional control i. These are controls that occur after the RNA polymerase has already started synthesizing the mRNA ii. Some mRNA molecules contain riboswitches, short RNA segments that can change their conformation when certain chemicals are present and therefore can control their own expression 1. There are no proteins involved in this type of regulation iii. Controlling translation 1. Repressor proteins can bind onto the 5’ cap in eucaryotic mRNA and regulate when the ribosomes can attach to carry out translation 2. RNA interference (RNAi) is one way organisms can control translation a. Many viruses have double stranded RNA (dsRNA) i. completely or just parts ii. As a defense against these viruses, cells have the ability to recognize dsRNA and trigger the RNAi response b. A nuclease named “dicer” recognizes and cuts dsRNA into small pieces called short interfering RNAs (siRNA) that are usually around 20 base pairs long i. This is a highly conserved nuclease found in many organisms 1. It probably evolved prior to the divergence of plants, animals, and fungi 2. Recently, a similar enzyme has been found in bacteria ii. siRNAs from dicer combine with a group of proteins to form a complex known as RISC (RNA-induced silencing complex) 1. The siRNA is denatured into a single strand a. In some systems there is preferential selection of strands but in most both an and are used 2. One of the key proteins is a protein called “slicer” iii. The RISC complex then uses the single stranded siRNA to bind to the complimentary mRNA iv. Slicer then cleaves the mRNA and the cell destroys the sliced transcripts c. Another type of double stranded RNA exists that is found naturally in many cells called microRNA (miRNA) i. These are expressed from non-coding regions of the genome and make natural double stranded RNA ii. miRNAs are also recognized by dicer and target other genes as a natural form of controlling gene expression without cleavage iii. miRNAs do not induce cleavage of the mRNA because they are not completely complimentary 1. Instead they are involved in translational repression Essential Cell Biology Chapter 8

Biotechnology Unit 3: DNA to Proteins 2. Specific type of miRNA called a small temporal RNA (stRNA) iv. Dicer interacts with the stRNA or other mRNA and then they enter the RISC complex and bind to mRNA but block translation instead of cutting v. There is imperfect recognition so slicing does not occur vi. This allows for the same miRNA to target several different genes or versions of genes

Essential Cell Biology Chapter 8