Microbiology 251 Study Guide
Chapter 6, 7, and 8
What RNA is the most abundant RNA in the cell, and which RNA polymerase is responsible for synthesizing it?
The most abundant RNA is the cell is rRNA. RNA polymerase I synthesizes rRNA.
What is responsible for forming the nucleolus? What happens there? The nucleolus is not membrane bound but it contains all components necessary for ribosomal production, which is its function. Some of these components are rRNA genes, precursor rRNAs, mature rRNAs, rRNAprocessing enzymes, snoRNPs, assembly factors, ribosomal proteins, and partly assembled ribosomes. These components are aggregated to make them more accessible for ribosome manufacture.
What are snoRNPs? What do they do?
snoRNPs are small nucleolar ribonucleoproteins that help to process and chemically modify rRNAs.
Three different types of RNA are involved in translation. What are they and what are their functions? What types of RNA Polymerase make each of them? Which RNA contains codons and which RNA contains anticodons? rRNA: ribosomal RNA; comprise ribosomes (along with ribosomal protein), catalyze protein synthesis; made by RNA polymerase I
tRNA: transfer RNA; charged with appropriate amino acid which is added to polypeptide chain; made by RNA polymerase III; contains anticodons mRNA: messenger RNA; carries genetic message into cytoplasm for translation; made by RNA polymerase II; contains codons
Why is the genetic code considered degenerate? If you want to learn more check out What are the types of gastropods?
The genetic code is considered degenerate because some amino acids are associated to more than one codon. For instance, AGC and UCA both are codons that translate to the amino acid serine.
Why is the reading frame important?
Reading frame is important because a shift could cause the addition of incorrect amino acids to the growing polypeptide. Different amino acids have different properties which would be applied in the new polypeptide.
What is the sequence of the initiator codon in mRNA? What is the sequence of the corresponding anticodon, written in the 5' to 3' direction? The initiator codon is 5’AUG3’ and it codes for the amino acid methionine. The anticodon would be 5’CAU3’.
What parts of an amino acid and the tRNA are involved in forming the covalent bond between these two molecules?
The carboxyl group of the amino acid and the 3’ end of the tRNA are involved in forming the covalent bond. If you want to learn more check out What interacts physically and chemically with one another and extracellular material to form defined tissues?
Which end of a polypeptide chain is encoded by information nearest the 5' end of a gene: N or C terminus?
The C terminus is nearer to the 5’ end because the gene will be read 3’ to 5’ and amino acids are added 5’ to 3’.
How does aminoacyltRNA synthase work? What molecules must aminoacyltRNA synthase recognize? What are the two Quality Control checks that it does to ensure that the right AA is charged on the right tRNA?
AminoacyltRNA synthetase uses energy in the form of ATP to add the appropriate amino acid to the 3’ end of a tRNA molecule. This enzyme must be able to recognize the correct amino acid, according to the anticodon sequence, and also incorrect amino acids to be able to accurately proofread. Aminoacyl tRNA synthetase has 2 sites for proofreading. The first, the synthesis site, recognizes the correct amino acid. The second, the editing site, recognizes only incorrect amino acids. If an incorrect amino acid makes it past the synthesis site, because of structural similarities with the correct amino acid, then it will be removed in the editing site where only incorrect amino acids are recognized. Don't forget about the age old question of What is the content of the sugar act of 1764?
Don't forget about the age old question of How many spinal nerves come off the cord?
Which part of an amino acid attacks the bond between an amino acid and a tRNA to form a peptide bond?
The carboxyl end of the previously added amino acid attacks the amino group of the amino acid still attached to the tRNA.
What are the different features of tRNAs that are important for them to function?
tRNA molecules have a clover leaf like folded configuration. This folding allows for base pairs on the same molecule to bind with other base pairs. This increases the stability of the tRNA molecule. This folded structure is also the ideal shape and size to be able to fit into a ribosome when a new amino acid is added to a polypeptide chain.
True or false: mRNAs are the only RNAs that are processed/modified in eukaryotes. Justify your response.
False. tRNA molecules undergo modifications that can include splicing or chemical modification. These chemical modifications include the addition of an alternate nucleotide. These alternate nucleotides can have functions in affecting the base pairing of the anticodon allowing for more accurate codon recognition or affecting the accuracy of the correctness of the attached amino acid. We also discuss several other topics like How does the movement of the myofibrils lead to muscle contraction?
What is the ribosome made of? Roughly how much does each component contribute?
A ribosome is made up of RNA and protein. About 2/3 of the ribosome is RNA and 1/3 is protein.
There are 4 RNA binding sites in a ribosome. What are they? What is found in each of them?
There is an mRNA binding site, an exit (E) site, an aminoacyltRNA (A) site, and a peptidyltRNA (P) site. An incoming mRNA binds in the mRNA site and the E site is where the tRNA without an associated amino acid (just added to polypeptide chain) is ejected. In the A site, a tRNA carrying the next amino acid to be added to the chain binds by forming base pairs with the codon positioned there by the mRNA. In the P site, the carboxyl group of the amino acid positioned there is released from tRNA and is joined to the amino acid in the A site via peptide bond.
What elongation factors are used in translation? What do they use to work? In prokaryotes, 2 elongation factors that are used are EFTu and EFG. In eukaryotes, 2 elongation factors that are used are EF1 and EF2. Elongation factors hydrolyze GTP to GDP to function.
Why is a ribosome called a ribozyme?
A ribosome is called a ribozyme because it consists of mostly RNA and has catalytic activity like an enzyme.
What part of the ribosome is responsible for catalyzing the peptidyl transferase reaction? If you want to learn more check out How is covering and lining epithelium categorized?
The P site in the large ribosomal subunit catalyzes the peptidyl transferase reaction and adds a new amino acid to the polypeptide chain.
How does the stop codon cause translation to stop?
No tRNA base pairs with a stop codon. A release factor protein, which is tRNA shaped, associates with the A site and causes hydrolysis of the polypeptide tRNA link. This reaction adds a water molecule to the end of the chain instead of an amino acid and the polypeptide is released from the ribosome.
What is meant by the terms poly and monocistronic?
Monocistronic mRNA codes for one protein per mRNA. This is unlike polycistronic mRNA which can code for multiple proteins per mRNA.
What is different about how eukaryotic ribosomes find the start codon versus the way a prokaryotic ribsosomes find the start codon? Eukaryotes have a 5’ cap that is recognized by a small ribosome subunit. After the small subunit associates with the cap, the small subunit can scan the mRNA sequence for the first AUG codon. In prokaryotes, there is no 5’ cap. Instead, each bacteria contains a specific ribosome binding site (ShineDelgarno sequence) that is located a few nucleotides upstream of the first AUG that forms a base pair with the small ribosomal subunit.
What does eIF2 do? How does it work?
eIF2 escorts the initiator tRNA into the P site. It is powered by GTP hydrolysis.
What role does the 5’ cap play in translation initiation? What protein recognizes the cap?
eIF4 recognizes the 5’ cap and recruits the small ribosomal subunit to begin translation. After the small subunit binds, it scans for the first AUG sequence and translation can begin.
When does the large subunit of the ribosome associate with the small subunit during translation?
The large subunit associates with the small subunit once the small subunit and the initiator tRNA have found the first AUG codon.
True or false: mRNAs are translated once and only once.
False. mRNAs are translated as much as the cell requires the finished protein product.
What factors assist protein folding in the cell (names of the class of proteins)? Do they use energy to work? How do they work? The molecular chaperones of the heat shock protein (hsp) 60 class assist protein folding by refolding misfolded proteins. There are 2 that work together the small subunit GroES and the large subunit GroEL. They break the bad folds and allow the correct ones to for inside the chaperone. They hydrolyze ATP to perform.
What are the possible fates of a misfolded protein?
A misfolded protein can be incorrectly folded but may get the chance to fold correctly with chaperones. If not, the misfolded protein is subject to degradation by a proteasome.
What is it attached to proteins to mark them for degradation? Enzymes mark proteins to be degraded with a small protein called ubiquitin to lysine residues.
What enzymes attach ubiquitin to proteins?
Ubiquitin is attached to proteins by a chain reaction of E1, E2, and E3 proteins.
True or false: only misfolded proteins are actively degraded in the cell. False. Proteins can be degraded based on the needs of the cell. If too much of a correctly folded protein is present, some of the protein will be degraded.
How are proteins targeted for degradation? How are they eliminated from the cell?
Proteins contain their own regulated degradation signals. They may be activated by phosphorylation, dissociation, or by destabilizing the N terminus.
What are the roles of the core and cap of the proteasome? The core of the proteasome contains the protease enzymes which break down proteins. The cap of the proteasome binds to proteins that have been marked for degradation.
What serves as degradation signals? Are they always active? If so, why? If not, why?
Degradation signals can be created from intracellular signals or signals from the environment. They are not always active because a cell may still be able to use the protein. It would not be able to complete its function when subjected to degradation.
Can you describe three ways ATP that is used to mediate the destruction of a protein?
ATP powers proteasomes which break down proteins with enzymes called proteases. ATP is used to attach ubiquitin proteins that are used as degradation signals for proteasomes. Finally, ATP is used to directly phosphorylate a protein indicating that it should be degraded.
What are the different steps in the control of gene expression in eukaryotes? Which occur in the cytoplasm and the nucleus? Steps of control of gene expression that occur in the nucleus are transcriptional control, RNA processing control and RNA transport and localization control. The other steps of control occur in the cytoplasm and they are translational control, mRNA degradation control, and protein activity control.
What does the term “motif” mean?
A motif is a significant sequence of nucleotides which gene regulatory proteins can recognize.
How do DNAbinding proteins recognize specific sequences? Does this binding disrupt the base pairing that holds complementary strands of DNA together?
DNA binding proteins recognize specific sequences based on the edges of the bases. These are the hydrogen groups, methyl groups, or H bond acceptors/donors present in the major/minor groove. Recognition of these molecules does not disrupt base pairing.
Explain why most DNAbinding proteins recognize specific sequences by making contact through the major groove rather than the minor groove of DNA
Most DNA binding proteins make contact with the major groove because it is larger and is more easily accessible. In addition, since it is larger, it contains more molecular features for the proteins to bind to.
What type of amino acid is usually enriched in the DNAbinding domain of proteins? Why?
Basic amino acids are enriched because the backbone of DNA has a negative charge. To more easily bind to DNA, these amino acids are used because they have a positive charge.
What is the secondary structure present in all of DNAbinding proteins we discussed in class that makes contact with the DNA?
Transcription regulators are present in all of the DNA binding proteins and make contact with the DNA.
What are the three advantages of dimerization of DNA binding proteins? Dimerization increases the length of the cis regulatory sequence, increases the affinity of the transcription regulator binding, and increases the specificity of the transcription regulator binding.
Bacterial promoters have operators and promoters. What's the difference
between these two types of sequences and what proteins recognize these two sequence elements?
The cis regulatory sequence is the operator and the promoter allows association of RNA polymerase. RNA polymerase binds at the promoter and begins transcription at the operator. Transcription factors recognize the promoter and the operator.
Lambda repressor functions as a repressor at one promoter and an activator at another promoter in bacteria? Explain how the same protein can have opposite activities at two promoters
Both repressors and promotes must bind a specific small molecule; therefore, they are structurally similar. They can function as both depending on the location of their cis regulatory sequence in relation to their promoter. Repressors would have promoters and cis regulatory sequences in the same location so the binding of transcription factors to the cis sequence would prohibit RNA polymerase from binding.
Describe how the tryptophan repressor regulates gene expression? The cis regulatory sequence for the tryptophan repressor is within the promoter. When a transcription factor binds to the sequence, as a result of high tryptophan levels in the cell, RNA polymerase in unable to bind to the promoter.
The lac operon and its regulation is a bacterial paradigm for how a series of on/off switches regulate the activity of this operon. Explain how the DNA binding activities of CAP and Lac repressor are regulated by glucose and lactose respectively to cause this operon to be active only when Glucose is low and lactose is high? What is the DNAbinding protein that serves to sense Glucose levels and what is the DNAbinding protein that serves to sense Lactose?
When glucose is absent in the cell, E. coli makes cAMP. This higher level of cAMP activates the CAP activator to try to initiate the transcription of genes that utilize different carbon sources such as lactose. This action only works if lactose is present in the cell. If lactose is not present, the Lac repressor will be bound to the sequence and won’t allow transcription by RNA polymerase. Therefore, only the absence of glucose and the presence of lactose will enable the lac operon to function.
What are the mechanisms that control the activity of gene regulatory proteins?
In eukaryotes, gene repression requires a co-repressor and gene activation requires a co-activator.
Where does a prokaryotic activator bind in relation to the promoter? How
does it differ from where a eukaryotic binds?
Prokaryotic activators bind adjacently to the promoter. This is unlike where a eukaryotic activator binds which can be long distances from the promoter to act at a distance.
Why is it said that eukaryotic activators and repressors work “at a distance”?
Eukaryotic repressors and activators can be effective even when they act many nucleotides away.
How do eukaryotic activators increase transcription? How do eukaryotic repressors decrease transcription?
Eukaryotes use coactivators and corepressors for each respective process. Activators increase transcription by attracting and positioning RNA polymerase at the promoter. Activators also assist by reorganizing the cell’s chromatin so that it is more accessible to RNA polymerase. Repressors can also lower the rate of transcription or turn it off completely by competitively binding with an activator, allowing an activator to bind but repressing its activity, interacting with transcription factors, or by altering the chromatin structure (either deacetylating or methylating.)
How do sequence specific activators, such as GAL4 in the example presented in class, differ from coactivators and corepressors? A sequence specific activators is directly associated with the mRNA whereas a coactivator or corepressor is associated with the activator and not a specific sequence of nucleotides.
How does chromatin affect the transcription of a gene?
The highly ordered structure of chromatin makes it difficult for transcription factors, activators in particular, to access the necessary genetic information.
Why must nucleosomes be moved or removed from promoters for genes to be expressed?
Nucleosomes need to be manipulated in order to access the promoter region which may be tightly wrapped. If the promoter region cannot be accessed, transcription will not occur.
How do ATPdependent chromatin remodelers work (at the level presented in lecture and in the textbook)?
Chromatin remodeling complexes work by hydrolyzing ATP to move nucleosomes away from promoter binding sites so that these sites are free for general transcription factors and RNA polymerase to bind.
What is the histone code? What chemical modification correlates with transcriptional activation?
The histone code is the covalent modification of histones and the impact it has on transcription. Methylation corresponds to repressing transcription and acetylation corresponds to activating transcription.
Explain how acetylation of histones can alter transcription? Histone acetylation allows the DNA to unpack from the histone. This unpacking makes the DNA loose and much easier to transcribe.
What are the differences between a chromatin remodeling complex and a histone modifying enzyme? How do they change chromatin structure at genes? What are HDACs?
Chromatin remodeling complexes operate on a physical level by moving nucleosomes. This is unlike a histone modifying enzyme which operates chemically by adding or taking away chemical groups on a histone. If a gene needs to be repressed, the chromatin structure is methylated. If the gene needs to be expressed, the chromatin is acetylated. Also, there are enzymes which remove the chemical groups described. Histone deacetylase or HDAC removes acetyl groups, instead of adding methyl groups, and represses transcription.
How do repressors work? Which mechanisms affect chromatin? Repressors can bind to activators or gene regulatory proteins to reduce transcription. They can also recruit histone deactylases or methyl transferases to further package the chromatin.
What is epigenetic regulation of transcription? Would a change in DNA sequence in the promoter be an example of epigenetic regulation? Epigenetic regulation of transcription involves the passing of a gene, either activated or repressed, to a daughter cell. A change in the DNA sequence of a promoter is not an example of this because the DNA sequence itself is not altered from parent cell to daughter cell. Modifications of the chromatin packaging are what are passed along.
What is epigenetic inheritance and describe three mechanisms by which this occurs?
Epigenetic inheritance is the ability of a daughter cell to retain the memory of a gene expression that was present in a parent cell. This can occur through a positive feedback loop by a transcription regulator, DNA methylation, and histone modification.
What is an advantage of having to splice a large premRNA into a smaller mature mRNA?
Starting with a large premRNA allows the same gene to code for different mRNAs depending on how it is spliced. This way, all cell types have this gene but only transcribe what they need.
What is similar about the mechanism of how alternative splicing is regulated in eukaryotes and how transcription is regulated in bacteria? In the simplest examples, alternative splicing can be thought of as off or on for a particular gene. This is similar to the off or on aspect of operons in bacterial transcription.
Suppose you have a premRNA with the following structure: Exon 1 Intron 1 Exon 2 Intron 2 Exon 3. Which exons and introns would be present in the spliced RNA if a splicing repressor bound over the 3’ splice site between intron 1 and exon 2?
Intron 1 Exon 2 Intron 2
How can alternative polyadenylation site utilization regulate the protein produced?
Differing polyadenylation sites can make very similar proteins that only differ in the amino acid sequences at the C terminus end.
What are the general functions of the 5’ UTR? The 3’UTR? The 3’UTR can contain a bound protein that localizes the mRNA. It can also decrease translation initiation by disrupting the communication between the 3’ end and the 5’ end. Also, the mRNA can be subjected to degradation if an endonuclease has cut out certain sequences in this area. In the 5’UTR translation can be repressed by the formation of hairpins in the mRNA or by the binding of a repressor protein. If the AUG start codon is within the hairpin, translation will not occur. Additionally, if a repressor protein is bound, translation will not occur.
How are mRNAs destroyed? What determines if it is destroyed faster or slower?
There are a class of RNAs called micro RNAs (miRNAs) that, after some processing, is assembled into a complex with a set of proteins to form an RNA induced silencing complex or RISC. This complex is a short string of nucleotides that binds to free mRNA molecules. If the base pairing between the two is extensive, the mRNA is destroyed quickly by exposing the mRNA’s polyA tail to exonucleases. If the base pairing is not extensive, the translation of the mRNA is repressed and it is sequestered from ribosome in Pbodies and eventually degraded.
What are the mechanisms that regulate translation of specific mRNAs? Where in the mRNA are the signals mediating this control often located? miRNAs regulate the translation of specific mRNAs because they have a nucleotide sequence that needs to match particular mRNAs. The better the base pairing, the more quickly the mRNA is degraded. The polyA tail is what is affected by miRNAs. They either immediately use exonucleases to degrade it or bind loosely to mRNA and repress translation until degradation can take place.
What are microRNAs (miRNAs)? How do they control gene expression? What two processes are regulated by miRNAs?
miRNAs are small RNA molecules that degrade mRNA in cells to control translation. They control gene expression by degrading mRNAs based upon how well they base pair with the mRNA strand. Translation and degradation are regulated by miRNAs.
How do you isolate different subcellular fractions? What physical property do you exploit when doing so?
Different subcellular fractions can be isolated using a centrifuge spinning at different speeds. Density is the property that is exploited in this process.
When using a centrifuge to separate components of the cell, which requires a greater centrifugal force to pellet, the ribosome or nucleus? The ribosome requires a greater centrifugal force because they are smaller and have a smaller density so more force needs to be applied.
What is the difference between ionexchange chromatography, gelfiltration chromatography, and affinity chromatography? If you attach an enzyme substrate to the surface of a column resin and use this to isolate a specific enzyme, which type of chromatography would this be called? In each case, a sample is poured into a column with special “beads”. These beads can have different properties put into them. For ion exchange chromatography, the beads are charged and the particles of the opposite charge are attracted to them. For gel filtration chromatography, the beads are porous and because of this, smaller molecules get trapped inside the beads and larger ones do not. Finally for affinity chromatography, the beads contain a substrate that has an affinity for something else, like a DNA sequence. The DNA sequence on the bead will trap particles with the complementary pair in the solution. The second part would be considered affinity chromatography because enzymes are highly specific for their substrates.
How do you elute the protein out from ion exchange chromatography?
To get the protein off of the beads, introduce something that is the same charge as the beads in higher concentration than the beads so that the protein has a higher chance of interacting with the substance.
What’s the relationship between the shape of a protein and how quickly it elutes from a size exclusion column?
The larger the protein, the more quickly it elutes from a size exclusion column. The smaller particles are trapped in the pores of the beads while the larger particles are too large to fit inside the beads and just travel past them.
What is the purpose of SDS and mercaptoethanol in the gel electrophoresis of proteins? On what basis does SDSPAGE separate proteins? SDS denatures proteins and coats the polypeptide chain so that it has a negative charge. This way, it’ll move through the gel based on its size and not its intrinsic charge. Mercaptoethanol breaks disulfide bonds between protein subunits so that each can move through the gel based on individual size. SDSPAGE separates proteins based on their size.
Why do proteins move through a gel during electrophoresis? Proteins move through the gel if they are negatively charged. How far these molecules get is determined by their size and shape.
What procedures are used to identify a protein in a gel?
The proteins are visible because of stains that are applied to them and can then be separated. The protein can then be determined using mass spectrometry.
Why would you want to attach an epitope tag to a protein you are studying?
Epitopes are recognized by highly specific antibodies that can then be used to purify the protein.
What is a western blot? Why would you use it?
Western blotting helps to identify specific proteins. This method involves dyed antibodies to determine which proteins are present. It is especially useful to access the amount of protein in a cell or to measure changes of protein concentration under various conditions.
What reagents do you need to run a western blot? What is a primary antibody? What is a secondary antibody? Why would you want to use both types?
Wash buffers and blocking solutions are necessary to increase contrast between the proteins and the background. A primary antibody recognizes the target protein but may not be directly detectable; thus, the introduction of a secondary
antibody (which also binds to the protein) is introduced. This secondary antibody is often tagged with some sort of marker, like fluorescent color.
What techniques are used to obtain high resolution structural models of proteins? What are some benefits and drawbacks (Pro’s and Con’s) to each of these procedures?
Techniques like Xray crystallography, Nuclear Magnetic Resonance (NMR), and CryoElectron Microscopy (CryoEM) are used to obtain structural models. Xray crystallography can be used with any size protein but often requires a large amount of it because a crystal must form with a repeating unit. NMR is limited to small proteins and there is no need to crystallize a repeating protein unit; however, this method uses heavy atoms instead which are costly. Finally, Cryo EM is limited to medium to large proteins and is becoming more popular with advancements in detectors used.
What information can Mass Spectrometry tell you about your ProteinOf Interest?
Mass spectrometry can determine the precise mass of the protein of interest. These values are very accurate, often with an error of less than one part in a million.
What is the difference between Monoclonal Antibodies and Polyclonal Antibodies?
Monoclonal antibodies are antibodies made from identical immune cells whereas polyclonal antibodies are antibodies made from different kinds of immune cells.
When comparing sequences of proteins, what does the identity and similarities scores tell you?
If these scores are similar, then the proteins in question likely have similar structure and function.