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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 __________________ stored in the 4 letter code of DNA direct the __________________ and __________________ of an entire organism II. The central dogma of molecular biology a. There are 2 general steps to the production of a __________________ from a __________________ i. Genes code for __________________ (__________________) ii. RNA codes for the __________________ (__________________) b. So, the flow of genetic information is from __________________ __________________ __________________ i. This is known as the __________________ of molecular biology c. Together, transcription and translation are the means by which cells __________________ their genetic information i. This is known as gene __________________ III. __________________ of DNA into RNA a. RNA is an __________________ produced from DNA in the process of protein __________________ i. RNA can have many forms and an incredibly wide range of __________________ within the cell b. Like DNA, RNA is a linear __________________ made up of four different __________________ linked together by phosphodiester bonds i. There are two major __________________ differences between DNA and RNA 1. The nucleotides in RNA are __________________ instead of deoxyribonucleotides a. They contain the 5-carbon sugar __________________ instead of deoxyribose 2. The bases involved in the ribonucleotides are A, C, G, and ____ (__________________) a. Uracil replaces __________________ and complimentarily binds with __________________ with 2 hydrogen bonds just like thymine ii. There is also a fairly major __________________ difference between DNA and RNA 1. RNA is __________________ stranded instead of being a double helix like DNA a. This allows RNA to __________________ into many different shapes to accomplish many different __________________ c. All RNA in a cell is made by __________________ i. There are several similarities between transcription and DNA __________________ 1. A small portion of the DNA double helix is opened up and __________________ to expose the bases of the DNA 2. One of the two strands acts as a __________________ and ribonucleotides are added using __________________ base-pairing 3. The new RNA molecule (called a __________________) grows one nucleotide at a time Essential Cell Biology Chapter 7 Biotechnology Unit 3: DNA to Proteins a. The enzyme responsible for reading the template and building the new RNA molecule is called __________________ and it reads from __________________ and builds from 5’ to 3’ like DNA polymerase i. RNA polymerase uses energy from ribonucleoside __________________ for energy just like DNA polymerase ii. Transcription does differ from DNA replication in some __________________ ways 1. The RNA transcript that is formed does not stay __________________ bonded to the template strand like DNA does a. The RNA ends up being __________________ stranded b. Just behind the region where nucleotides are being added, the RNA transcript is __________________ and the DNA double helix __________________ 2. Only one side of the DNA molecule acts a __________________ during transcription a. Both sides can be templates for different genes at different __________________ on the chromosome however b. The __________________ or __________________ strand is the strand that is __________________ by the RNA polymerase c. The other strand is called the __________________ or __________________ strand and is the __________________ for the RNA molecule 3. RNA molecules are only transcribed from __________________ regions of DNA a. This means that transcription only produces a __________________ segment of not more than a few __________________ base-pairs and not an entire DNA molecule 4. Transcription of the same gene can occur __________________ by several different RNA polymerases a. Because the transcripts are released almost __________________, transcription can start over shortly after the RNA polymerase has moved on from the starting point b. It takes approximately __________________ to transcribe a molecule of RNA from a __________________ sized gene of 1500 nucleotide pairs i. As many as 15 RNA polymerases can __________________ be transcribing this gene at any one give time ii. This means over __________________ can be synthesized in a hour 5. RNA polymerase is very similar in its action as DNA polymerase but there are a couple key __________________ a. RNA polymerase polymerizes __________________ instead of deoxyribonucleotides b. RNA polymerase does not require RNA __________________ i. Because RNA does not __________________ store genetic information, the necessity of __________________ in transcription is lower than in DNA 1. RNA polymerases make approximately 1 mistake in every __________________ compared to the rate of one mistake in 107 nucleotides by DNA polymerase iii. Several types of RNA are __________________ in cells 1. The vast majority of genes in a cell’s DNA code for the amino acid sequence of __________________ Essential Cell Biology Chapter 7 Biotechnology Unit 3: DNA to Proteins a. The RNA molecules that are copied from these genes are called __________________ RNA (__________________) i. In __________________, each mRNA carries information from just one gene, coding for just one protein ii. In __________________, 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 __________________ itself a. These nonmessenger RNAs can serve a __________________, __________________, and __________________ components of cells and they are important to the translation of proteins and the regulation of gene expression b. __________________ RNA (__________________) forms the central core of the ribosomes where mRNA is translated into proteins c. __________________ RNA (__________________) for the adaptors that hold the amino acids in place on the ribosome so they can be incorporated into the protein d. __________________ (__________________) serve as regulators of eukaryotic gene expression d. __________________ in DNA tell RNA polymerase where to start and finish i. Because of the fact that not all of the DNA of an organism __________________ for proteins and because not all of the genes are __________________ in all cells, there has to be a __________________ to tell RNA polymerase where to begin transcription ii. The beginning of a gene must be __________________ in order for transcription to begin iii. This process is different in __________________ and __________________ 1. In procaryotes, when RNA polymerase collides with a DNA molecule it attaches itself very __________________ and moves along the DNA until it finds a specific region called a __________________ a. At the promoter, the RNA polymerase binds __________________ and opens up the double helix in front of itself i. Promoter regions are __________________ 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 __________________ strand and producing the __________________ RNA transcript until it reaches another specific region called the __________________ or stop site i. At the terminator site, the RNA polymerase __________________ from the DNA double helix c. The procaryotic RNA polymerase has a special subunit called a __________________ that helps to identify the promoter region i. Once the promoter is identified, the sigma factor __________________ itself from the RNA polymerase until transcription is __________________ and the polymerase has disengaged 2. In eucaryotes, the process is much more __________________ a. Unlike procaryotes, eucaryotes have __________________ different RNA polymerases i. RNA polymerase I and RNA polymerase III are responsible for transcribing __________________, __________________, and __________________ Essential Cell Biology Chapter 7 Biotechnology Unit 3: DNA to Proteins ii. RNA polymerase II is responsible for transcribing __________________ 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 __________________ which assemble at the __________________ before transcription can occur i. A group of __________________ __________________ __________________ 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 __________________ ii. Once the RNA polymerase II is bound to the transcription factor complex, it is __________________ 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 __________________ at another promoter region c. The __________________ 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 __________________ sequences for individual genes d. Finally, the __________________ of the eukaryotic chromosomes adds a level of complexity to transcription e. __________________ of transcripts i. In prokaryotes, the DNA is already in the __________________ and exposed to ribosomes 1. __________________ 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 __________________ inside the nucleus and is not exposed to the ribosomes until it leaves 1. Eucaryotic transcripts go through several steps of RNA __________________ before they can leave the nucleus a. These steps take place while transcription is occurring because the enzymes are attached to the __________________ of the RNA polymerase b. RNA __________________ involves adding methylated __________________ to the 5’ end of the transcript c. __________________ involves cutting a small portion off of the 3’ end of the transcript and then adds a few hundred __________________ creating a poly-A tail d. Both RNA capping and polyadenylation are thought to add __________________ and to help with the __________________ of the mRNA out of the nucleus 2. Most eukaryotic genes are __________________ with noncoding regions that need to be __________________ from the transcript before translation can occur a. The noncoding regions are called __________________ and the coding regions are called __________________ i. The introns are usually much __________________ than the exons Essential Cell Biology Chapter 7 IV. Biotechnology Unit 3: DNA to Proteins b. The __________________ length of a gene is transcribed including both introns and exons c. The introns need to be removed by RNA __________________ i. There are sequences within the introns that __________________ 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 (__________________) that combine with proteins to form small nuclear ribonucleoprotein particles (__________________) (pronounced “snurps”) iii. The cutting follows what is known as a __________________ cut 3. __________________ splicing provides the ability for one mRNA to be used to produce several different __________________ a. Splicing the RNA __________________ (keeping different exons) provides many different functional mRNA molecules b. It is estimated that ________ of human genes undergo alternative splicing iii. Once RNA processing and splicing is complete, the mRNA must __________________ the nucleus to join with ribosomes to be translated into proteins __________________ is the process of converting the RNA sequence into a __________________ a. Translation involves a much more __________________ change than transcription i. The “__________________” used to carry the code in DNA and RNA is based on 5 (but only 4 at a time) __________________ ii. The “__________________” used to build proteins is composed of 20 __________________ 1. There cannot be a one to one relationship between RNA and proteins so we need what is referred to as the __________________ __________________ that is identical in nearly all life forms a. The mRNA sequence is read in consecutive groups of 3 nucleotides called __________________ i. There are 4x4x4 = __________________ for codon combinations ii. Because there are only 20 amino acids, there is a built in __________________ meaning come amino acids are coded for by more than one codon b. This allows for three unique __________________ __________________ depending on where the reading begins i. There is a __________________ start codon that identifies the proper reading frame for each protein b. The rapid __________________ of mRNA into a protein requires a molecular __________________ that moves along the RNA, captures and holds tRNA molecules, and __________________ links the incoming amino acids i. __________________ RNA (tRNAs) molecules match amino acids to the proper mRNA codons 1. tRNAs are molecules roughly 80 nucleotides long that act as __________________ to bring in the correct amino acid that is coded for by each codon a. Each tRNA contains a region known as the __________________ which is complimentary to the codon on the mRNA b. The 3’ end of the tRNA holds a specific __________________ molecule c. tRNA molecules are said to be __________________ when the amino acid is attached Essential Cell Biology Chapter 7 Biotechnology Unit 3: DNA to Proteins i. This is done by enzymes called aminoacyl-tRNA __________________ ii. Each amino acid has a __________________ synthetase that is responsible for connecting that amino acid to the tRNA molecules with the proper anticodons ii. __________________ are the protein manufacturing machine 1. They are made of over 50 different __________________ and several __________________ RNA (rRNA) molecules 2. Typical cells have __________________ of individual ribosomes a. Both eucaryotic and procaryotic ribosomes are similar and are made up of a __________________ subunit and a __________________ subunit i. The small subunit pairs the __________________ to the mRNA codons ii. The large subunit links the __________________ into the polypeptide chain b. The ribosome attaches near the __________________ of the mRNA and then pulls the mRNA through while it __________________ the appropriate amino acids i. Eucaryotic ribosomes can add about __________________ per second ii. Procaryotic ribosomes can combine up to __________________ per second 3. Along with the binding site for the mRNA, the ribosome has __________________ sites for tRNAs a. The __________________ is where the new tRNA comes in and binds to the anticodon and brings in the new amino acid b. The __________________ is where the previous tRNA is held until the new amino acid can be linked onto the chain c. The __________________ holds the tRNA until it is ejected d. The mRNA moves through these sites __________________ until the polypeptide chain is complete 4. Codons in mRNA signal where to __________ and _________ protein synthesis a. Translation always begins with the codon __________________ i. The __________________ tRNA carries the amino acid methionine and attaches to the AUG codon ii. This initiator tRNA is different from the normal __________________ carrying tRNA iii. The __________________ ribosomal subunit attaches to the 5’end of them RNA (signaled by the 5’ cap) and then __________________ along until it finds an AUG b. It then reads along adding all the necessary amino acids until it reaches a __________________ codon i. __________, __________, and __________ are the stop codons ii. Stop codons do not correspond to a tRNA and instead signal the ribosome to stop __________________ iii. __________________ factors (proteins) bind to the stop codon in the A-site and cause the __________________ and the __________________ to be released from the mRNA Essential Cell Biology Chapter 7 Biotechnology Unit 3: DNA to Proteins Essential Cell Biology Chapter 7 Biotechnology Unit 3: DNA to Proteins 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 __________________ for that organism but not all of the __________________ are needed within the cell a. Different cell types produce different sets of __________________ b. The __________________ of each protein that the cell needs is different at different times c. The control of gene __________________ is how the cell deals with these issues i. Cells can change the expression of genes in response to __________________ signals II. Gene expression can be __________________ at many different levels or step within the __________________ from DNA to RNA to protein a. __________________ control i. Transcription is controlled by __________________ binding to __________________ DNA sequences 1. Remember that in order for transcription to begin, RNA __________________ must attach and __________________ itself at the promoter region of the gene 2. The simplest regulatory sequences are only __________________ long a. These are most common in __________________ 3. In eucaryotes, the regulatory sequences can be up to __________________ nucleotide pairs and can interact with a wide variety of signals 4. Transcription __________________ are proteins that bind to specific regulatory DNA sequences to act as a __________________ for the control of transcription a. Different proteins recognize different __________________ based on the shape of the protein ii. In procaryotes, __________________ control the expression of groups of genes 1. There is a region at the start of the operon called the __________________ which is where transcription of the entire group of genes will begin 2. Operators are controlled by __________________ and __________________ a. A repressor is a __________________ that when bound to the operator does not allow transcription to begin by blocking the __________________ of RNA polymerase b. An activator is a protein that allows transcription to occur by allowing for __________________ attachment of the DNA polymerase c. Some operons use __________________ repressors and activators 3. Operon __________________ a. __________________ operon i. This operon uses a __________________ and it controls a group of five genes that are necessary for the production of the amino acid tryptophan ii. When tryptophan is in __________________ in the cell, it can bind to the repressor protein which will then bind to the operon and __________________ 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 __________________ for the production of tryptophan Essential Cell Biology Chapter 8 Biotechnology Unit 3: DNA to Proteins b. Lac operon i. This operon uses both a __________________ and an __________________ to control the transcription of several genes used to break down __________________ which is a food source for bacteria ii. __________________ 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 __________________ 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 __________________ is present iv. The only time the Lac operon is active, is when both the activator is bound (__________________ not present) and the repressor is not bound (__________________ is present) Essential Cell Biology Chapter 8 Biotechnology Unit 3: DNA to Proteins iii. __________________ also use transcription regulators (both repressors and activators) but they do not use __________________ to control groups of genes 1. These sites are called __________________ 2. Eucaryotic transcription regulators can work from a __________________ a. The binding of activators or repressors in eucaryotes does not need to be __________________ to the gene of interest or even be __________________ from it b. In many instances the transcription regulators are long distances away but there is a __________________ that occurs to allow for the initiation of transcription b. __________________ -transcriptional control i. These are controls that occur __________________ the RNA polymerase has already started synthesizing the mRNA ii. Some mRNA molecules contain __________________, short RNA segments that can change their conformation when certain chemicals are present and therefore can control their own expression 1. There are no __________________ involved in this type of regulation iii. Controlling __________________ 1. Repressor proteins can bind onto the __________________ in eucaryotic mRNA and regulate when the ribosomes can attach to carry out translation 2. RNA __________________ (RNAi) is one way organisms can control translation a. Many __________________ have double stranded RNA (dsRNA) i. completely or just parts ii. As a defense against these viruses, cells have the ability to __________________ dsRNA and trigger the RNAi response b. A nuclease named “__________________” recognizes and cuts dsRNA into small pieces called __________________ RNAs (siRNA) that are usually around 20 base pairs long i. This is a highly conserved nuclease found in __________________ organisms 1. It probably evolved __________________ to the divergence of plants, animals, and fungi 2. Recently, a similar enzyme has been found in __________________ ii. siRNAs from dicer combine with a group of proteins to form a complex known as __________________ (RNA-induced silencing complex) 1. The siRNA is __________________ into a single strand a. In some systems there is __________________ selection of strands but in most both an and are used 2. One of the key proteins is a protein called “__________________” iii. The RISC complex then uses the single stranded siRNA to bind to the __________________ mRNA iv. Slicer then __________________ the mRNA and the cell __________________ the sliced transcripts c. Another type of double stranded RNA exists that is found naturally in many cells called __________________ (miRNA) i. These are expressed from __________________ regions of the genome and make natural double stranded RNA Essential Cell Biology Chapter 8 ii. iii. iv. v. vi. Biotechnology Unit 3: DNA to Proteins miRNAs are also recognized by dicer and target other genes as a __________________ form of controlling gene expression without cleavage miRNAs do not induce cleavage of the mRNA because they are not __________________ complimentary 1. Instead they are involved in translational __________________ 2. Specific type of miRNA called a small temporal RNA (stRNA) Dicer interacts with the stRNA or other mRNA and then they enter the RISC complex and bind to mRNA but __________________ translation instead of cutting There is __________________ recognition so slicing does not occur This allows for the same miRNA to target several different __________________ or __________________ of genes Essential Cell Biology Chapter 8