MIBO 2500 Exam 3

The Role of Ribosomes

- A ribosome serves as a translation "machine" that strings amino acids together to make a polypeptide. It does this by aligning two amino acids and catalyzing the formation of a peptide bond between them. - Ribosomes also locate key punctuation sequences on the mRNA molecule, such as the points at which synthesis of a particular protein should start and stop

proofreading

- immediately correcting nucleotide during synthesis - During DNA synthesis - What enzyme could do this? · DNA polymerase

latent TB infection (LTBI)

- pts not sick because the organisms are not active. - they are asymptomatic and can NOT spread the disease -drug therapy help to prevent an infection from developing into the active disease. -Drug therapy for LTBI is only one drug instead of a cocktail of drugs (INH) · Siege strategy works 90% of the time - After a while the body won't be able to fight the infection and the tubercle will not hold, could be for numerous reasons like age · Foamy macrophage starts to break down into caseum and the tubercle will crack and the infection has lived while the macrophage has died and so the infection will begin to attack the body

At the origin of replication:

1. Helicase unwinds dsDNA 2. primase makes RNA primer to help polymerase 3. DNA polymerases adds nucleotides in the 5' to 3' direction onto growing strand

List four differences between eukaryotic and prokaryotic gene expression.

1. In prokaryotic cells, transcription and translation occur almost simultaneously. Meanwhile, in eukaryotic cells the processes of transcription and translation are physically separated by the nuclear membrane; transcription occurs only within the nucleus, and translation occurs only outside the nucleus in the cytoplasm. 2. Eukaryotic genes are split into exons and introns; in bacteria, genes are almost never split. 3. The mRNA in eukaryotic cells must be transported out of the nucleus before it can be translated in the cytoplasm. Thus, unlike in prokaryotes, the same mRNA molecule cannot be synthesized and translated at the same time or even in the same cellular location. 4. In eukaryotes, one mRNA = one protein. (in bacteria, one mRNA can be polycistronic, or code for several proteins).

Base substitution in a protein-encoding gene can lead to three possible mutation outcomes:

1. Synonymous mutation 2. Missense mutation 3. Nonsense mutation

Only ___ cells needed for an infection of TB

10

- Suggested regimen: latent TB

2 drugs taken weekly for 3 months

base analogs

5-bromouracil pairs with wrong base causing point mutation

each nucleotide consists of a

5-carbon sugar (deoxyribose), a phosphate group, and one of four different nucleobases (A, T, G, or C)

synonymous mutation (silent mutation)

A base pair substitution that does not change the amino acid that a codon normally produces the outcome is not always silent because the change can affect the efficiency of translation and other processes

lagging strand

A discontinuously synthesized DNA strand that elongates by means of Okazaki fragments, each synthesized in a 5' to 3' direction away from the replication fork.

DNA ligase

A linking enzyme essential for DNA replication; catalyzes the covalent bonding of the 3' end of a new DNA fragment to the 5' end of a growing chain.

Nonsense mutation

A mutation that changes an amino acid codon to one of the three stop codons, resulting in a shorter and usually nonfunctional protein. Any mutation that totally inactivates the gene is termed a null or knockout mutation.

Missense mutation

A point mutation in which a codon that specifies an amino acid is mutated into a codon that specifies a different amino acid. In many cases, cells with a missense mutation grow slowly because the encoded protein does not function as well as normal.

activator

A protein that binds to DNA and stimulates transcription of a specific gene.

repressor

A protein that binds to an operator and physically blocks RNA polymerase from binding to a promoter site

Codon

A specific sequence of three adjacent bases on a strand of DNA or RNA that provides genetic code information for a particular amino acid

inducer

A specific small molecule that inactivates the repressor in an operon.

monocistronic

A transcript that carries one gene

DNA polymerase

An enzyme that catalyzes the formation of the DNA molecule.

Helicase

An enzyme that untwists the double helix of DNA at the replication forks.

Tuberculosis

An infectious disease that may affect almost all tissues of the body, especially the lungs

The Role of mRNA

An mRNA molecule is a temporary copy of the information in DNA; it carries encoded instructions for synthesis of a specific protein, or in the case of a polycistronic message, a specific group of proteins

chemical mutagens

Chemicals that cause genetic mutations -- alkylating agents change purine and pyrimidine structure to cause point mutations

plasmids

Circular DNA molecules that can replicate independently of the main chromosomes of bacteria

Topoisomerase

corrects "overwinding" ahead of replication forks by breaking, swiveling, and rejoining DNA strands

identify the main method of transmission and pathogenesis for tuberculosis

coughing, sneezing, etc. droplets in the air from active infected TB patients

A fundamental aspect of the regulation is the cell's ability to quickly

destroy mRNA—within minutes of being produced, transcripts are degraded by cellular enzymes. This provides cells with a means to control gene expression.

Rifampin

inhibit RNA polymerase (for what cellular process?)

Isoniazid

inhibit mycolic acid synthesis

Ethambutol

inhibits other cell wall components

The operator

is a negative regulatory site bound by the lac repressor protein. The operator overlaps with the promoter, and when the lacrepressor is bound, RNA polymerase cannot bind to the promoter and start transcription.

The CAP binding site

is a positive regulatory site that is bound by catabolite activator protein (CAP). When CAP is bound to this site, it promotes transcription by helping RNA polymerase bind to the promoter

The promoter

is the binding site for RNA polymerase, the enzyme that performs transcription.

Signal transduction

is the process that transmits information from outside a cell to the inside. It allows cells to monitor and react to environmental conditions

bidirectional replication means

it proceeds in both directions from a specific starting point called the origin of replication

enzymes calles primases

synthesize short stretches of RNA complementary to the exposed templates. These small fragments, called primers, are important in the steps of replication

Primase

synthesizes RNA primer

base substitutions create

point mutations

a gene encodes a

product (called the gene product), most commonly a protein

anti-sigma factors

proteins that bind to sigma factors and inhibit their function

spontaneous mutations

random change in the DNA due to errors in replication/base substitutions that occur without known cause are random genetic changes that result from normal cell processes and are passed on to a cell's progeny - DNA polymerase can actually correct its mistake through proofreading

if it is then turned "off" the number of transcripts will

rapidly decline

Alternative Sigma Factors

recognize different sets of promoters to control expression of specific groups of genes

induced mutations

refers to those mutations in the DNA resulting from exposure to toxic chemicals or to radiation such as chemical mutagens base analogs intercalating agents UV irradiation X-rays

frameshift mutation

removal or addition of nucleotides, mutation that shifts the "reading" frame of the genetic message by inserting or deleting a nucleotide, stop codon may be created

RNA primer

short piece of RNA needed for DNA polymerase to start

The part of RNA polymerase that recognizes the promoter is a loosely attached subunit called

sigma factor

the plus strand

strand of DNA complementary to the one that serves as the template for RNA synthesis; the nucleotide sequence of the RNA molecule is the same as this strand, except it has uracil rather than thymine

Pyrazinamide

supposedly interferes with ribosome repair

Describe the process of transcription, focusing on the role of RNA polymerase, sigma factors, promoters, and terminators.

- DNA to mRNA - occurs inside the nucleus - sigma factors recognize promoters for genes that need to be expressed during routine growth conditions and indicates to RNA polymerase - *RNA polymerase* is used to create a new sequence of nucleotides that will become the messenger RNA, which it knows where to begin because it binds to the *promotor* region. - The strands are then able to separate (RNA polymerase separates the strand AND is able to code for the RNA, can only add nucleotides on the 3' end, so encodes in the 5' to 3' direction) (remember it codes for uracil instead of thymine) - a terminator sequence will be reached and and hairpin loop formed by RNA will indicate to the RNA polymerase to let go

Identify TB symptoms and complications such as LTBI

- Immune system lays siege, forms tubercle- latent TB infection (LTBI) · Siege strategy works 90% of the time - After a while the body won't be able to fight the infection and the tubercle will not hold, could be for numerous reasons like age · Foamy macrophage starts to break down into caseum and the tubercle will crack and the infection has lived while the macrophage has died and so the infection will begin to attack the body

Predict how spontaneous mutations that induce substitutions or frameshifts can enable the pathogen to resist the effect of an antimicrobial

- M. tuberculosis exhibits spontaneous chromosomal mutations - Mutations in RNA polymerase weaken binding affinity of rifampin, mutations alter binding site - pncA gene encodes pyrazinamide which converts pyrazinamide to its active form, pyrazinoic acid. Mutations in gene accounts for majority of resistant M. Tuberculosis

mRNA, tRNA, rRNA

- Most genes encode proteins and are transcribed into mRNA (messenger RNA) (The information encrypted in mRNA is decoded according to the genetic code, which correlates each set of 3 nucleotides to a particular amino acid) - The gene for rRNA (ribosomal RNA) and tRNA (transfer RNA) are never translated into proteins; instead, RNA molecules themselves are the final products, and each type plays a different but critical role in protein synthesis

Transposons

- The gene into which a transposon jumps is inactivated by the event, an outcome called insertional inactivation, meaning that the inserted DNA disrupts the function of the gene. Most transposons contain transcriptional terminators, so the expression of downstream genes in the same operon will stop as well. - Transposons, or jumping genes, are pieces of DNA that can move from one location to another in a cell's genome, a process called transposition - Transposons can be introduced intentionally into a cell in order to generate mutations. The transposon, which cannot replicate on its own because it lacks an origin of replication, inserts into the cell's genome. This generally inactivates the gene into which it inserts.

Using the lac operon, explain the roles of an inducer, repressor and activators.

- The lac operon is an operon, or group of genes with a single promoter (transcribed as a single mRNA). The genes in the operon encode proteins that allow the bacteria to use lactose as an energy source. - if lactose is the only sugar available, the E. coli will go right ahead and use it as an energy source (prefers glucose) - levels of lactose and glucose are detected, and two regulatory proteins are involved: One, the lac *repressor*, acts as a lactose sensor. The other, catabolite *activator* protein (CAP), acts as a glucose sensor. (These proteins bind to the DNA of the lac operon and regulate its transcription based on lactose and glucose levels) - The lac repressor is a protein that represses (inhibits) transcription of the lacoperon. It does this by binding to the operator, which partially overlaps with the promoter. When bound, the lac repressor gets in RNA polymerase's way and keeps it from transcribing the operon. When lactose is not available, the lac repressor binds tightly to the operator, preventing transcription by RNA polymerase. However, when lactose is present, Allolactose binds to the lac repressor and causes it to lose its ability to bind DNA. It floats off the operator, clearing the way for RNA polymerase to transcribe the operon. - Allolactose is an example of an *inducer*, a small molecule that triggers expression of a gene or operon. - CAP binds to a region of DNA just before the lac operon promoter and helps RNA polymerase attach to the promoter, driving high levels of transcription. (it's regulated by a small molecule called cyclic AMP (cAMP))

Translation termination

- The process of translation terminates when the ribosome reaches a stop codon, a codon not recognized by a tRNA. - At this point, enzymes free the polypeptide by breaking the covalent bond that joins it to the tRNA. - The ribosome falls off the mRNA, dissociating into its two component subunits (30S and 50S). the subunits can then be reused to initiate translation at other sites

Mismatch repair

- after DNA synthesis - Endonucleases to cut and tri away mistake - DNA polymerase to make new strand - DNA ligase to seal - Occurs after proofreading fails to work

Prokaryote DNA replication is

- bidirectional - semiconservative - begins at origin of replication for both chromosome and plasmid

Describe the process of translation, focusing on the role of mRNA, ribosomes - with the corresponding Psite, A-site, and E-site, rRNA, tRNA, and codons.

- going from the mRNA created in transcription to a protein, occurs in the cytoplasm - ribosome is made of rRNA - ribosome begins to assemble at the ribosome-binding site, joined by initiating tRNA (mRNA helps direct which are allowed in and which are not) that carries a chemically altered form of the amino acid - As the ribosome assembles, that initiating tRNA binds to the start codon after the ribosome-binding site and occupies a ribosomal region called the P-site. Thus, the initiating tRNA helps position the assembled ribosome relative to the first AUG, which is important because the position determines the reading frame used for translating the remainder of that polypeptide. - The ribosome advances a distance of one codon, a process called translocation, moving along the mRNA in a 5' to 3' direction. As this happens, the uncharged initiating tRNA is released through the E-site to be recycled. - The remaining tRNA, which now carries both amino acids, occupies the P-site. - A tRNA that recognizes the codon in the A-site then quickly attaches there. Soon, the amino acid chain carried by the tRNA now in the P-site will be transferred to the amino acid carried by the tRNA that just entered the A-site - The process of translation terminates when the ribosome reaches a stop codon, a codon not recognized by a tRNA. - At this point, enzymes free the polypeptide by breaking the covalent bond that joins it to the tRNA. - The ribosome falls off the mRNA, dissociating into its two component subunits (30S and 50S). the subunits can then be reused to initiate translation at other sites (A-site is where the appropriate tRNA initially binds to an amino acid, p-site is where the polypeptide chain begins to form, E-site is the site of exit)

using the following terms, describe the DNA replication process (playing the ebook video on mute can be helpful as well): origin of replication, DNA gyrase, helicase, primase, primers, DNA polymerases, 5' to 3', leading strand, lagging strand, Okazaki fragments, DNA ligase

DNA replication begins at the *origin* which is identified by certain DNA sequences. DNA has two strands, and *helicase* (also "DNA gyrase* is a type of Topoisomerase that reduces the strain of unwinding) (beginning at the origin) is used to "unzip" these strands. While "unzipping" it breaks through hydrogen bonds between the DNA bases together. You don't want these strands to come back together SO SSB proteins (single stranded binging proteins) bind to the DNA strands to keep them separated. *DNA polymerase* is the "builder". This enzyme replicates DNA molecules in order to build a new strand of DNA. *Primase* is the "initializer". DNA polymerase cannot figure out where to begin working on a DNA strand on its own, so primate makes *primers* so that DNA polymerase can begin "building". *Ligase* is the "gluer". It helps "glue" DNA fragments together. Remember DNA strands run either *5' to 3'* or 3' to 5', and this determines direction. DNA polymerase builds the new strand in the *5' to 3' direction* (this means it moves along the old, template strand in the 3' to 5' direction. The *leading strand* contains the strand that is building the new strand in the 5' to 3' direction on the 3' to 5' old strand. The "lagging strand" runs the new strand in the 5' to 3' direction on the 5' to 3' old template strand, and is called lagging because it is constantly having to catch up due to running in the opposite direction of where the unwinding is occurring. This creates *Okazaki Fragments*, and ligase has to glue together the gaps in between these fragments.

the genome of all cells is composed of _______, but some viruses have an ________ genome

DNA; RNA

Explain how nucleotide errors by DNA polymerase can be repaired.

During proofreading, DNA polymerase enzymes recognize this and replace the incorrectly inserted nucleotide so that replication can continue. Proofreading fixes about 99% of these types of errors, but that's still not good enough for normal cell functioning. Proofreading by DNA polymerase corrects errors during replication. Some errors are not corrected during replication, but are instead corrected after replication is completed; this type of repair is known as mismatch repair

DNA gyrase

Enzyme that temporarily breaks the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix

two-component regulatory system

Mechanism of gene regulation utilizing a sensor and a response regulator

Explain why gene expression is so important to a cell.

Gene regulation is important, because DNA is important for coding proteins and other products. These products have very special instructions and if given incorrectly could disrupt the process of an organism. The method of gene regulations helps to determine which genes are "turned on" or "turned off" which assists in the expression of genes when needed. EX: you can have regulatory proteins that bind to DNA that assist RNA polymerase in increasing the rate of transcription. OR you can have proteins that decrease the rate of transcription to the point that it may not be transcribed at all. It can also help us better understand diseases.

Testing for TB:

Injection of purified protein from bacteria, raised bump indicates potential positive result

Determine how certain antimicrobial activities kill M. tuberculosis by targeting replication or transcriptional activities

M. tuberculosis and other mycobacterial species are naturally resistant to macrolides and lincosamides. These antibiotics stop the growth of bacterial cells through inhibitory action on the protein synthetic machinery.

Identify TB symptoms and complications such as MDR-TB

Multidrug resistant strain of TB (MDR-TB): - M. tuberculosis exhibits spontaneous chromosomal mutations - Mutations in RNA polymerase weaken binding affinity of rifampin, mutations alter binding site - pncA gene encodes pyrazinamide which converts pyrazinamide to its active form, pyrazinoic acid. Mutations in gene accounts for majority of resistant M. Tuberculosis

Okazaki fragments

Small fragments of DNA produced on the lagging strand during DNA replication, joined later by DNA ligase to form a complete strand.

minus strand

The DNA strand that serves as the template for transcription

leading strand

The new continuous complementary DNA strand synthesized along the template strand in the mandatory 5' to 3' direction.

Compare the damaging effects on DNA of base analogs, intercalating agents, UV light and X rays.

These base analogs induce mutations because they often have different base-pairing rules than the bases they replace. Intercalating agents, such as acridine, introduce atypical spacing between base pairs, resulting in DNA polymerase introducing either a deletion or an insertion, leading to a potential frameshift mutation.There are a large number of chemicals that act as intercalating agents, can mutate DNA, and are carcinogenic (can cause cancer). Many of these are also used to treat cancer, as they preferentially kill actively dividing cells. UVB light causes thymine base pairs next to each other in genetic sequences to bond together into pyrimidine dimers, a disruption in the strand, which reproductive enzymes cannot copy. It causes sunburn and it triggers the production of melanin. Other names for the "direct DNA damage" are: thymine dimers. X-rays can directly affect a DNA molecule in one of three ways: Changing the chemical structure of the bases; Breaking the sugar-phosphate backbone; or. Breaking the hydrogen bonds connecting the base pairs.

Correlate the current approaches to TB treatment and monitoring to the number of recent TB cases in the US

To combat growing resistance: · TB Direct Observed Therapy Short-course (DOTS), healthcare workers administer and watch patients as they take their medication - TB rates in US is low, the overall US TB rate for 2019 was 2.7 cases per 100,000 persons - TB remains one of top 10 causes of death worldwide. According to WHO, 1.5 million people died worldwide by TB. 7 million people receive record levels of lifesaving TB treatment but 3 million still miss out

Explain why transcription and translation can occur simultaneously in prokaryotes and not eukaryotes

Transcription and translation are coupled in prokaryotes, because they don't have a nucleus. In eukaryotic cells, transcription happens inside the nucleus and translation can't happen until the mRNA is transported out into the cytoplasm. But in prokaryotes, everything is happening in the cytoplasm so as soon as the mRNA molecule starts to be made, there are ribosome ready to join and start making proteins

Summarize how the two-component system allows cells to adapt to environmental changes.

Two-component regulatory systems (TCRS) are important mediators of signal transduction that enable bacteria to detect physical and/or chemical changes and then relay this signal through the cytoplasm to the bacterial nucleoid, where modulation of gene expression occurs.

During transcription, translation can occur simultaneously Could replication also occur at the same time? Why is this possible in prokaryotes and not eukaryotes?

Yes, because it is at different location 1. Eukaryotic must move mRNA from nucleus to cytoplasm 2. Eukaryotic genes carry introns and exons, introns have to be spliced out, prokaryotes don't have introns can immediately translate mRNA

operon

a group of genes that operate together

origin of replication

a particular sequence in a genome at which replication is initiated

Mutations

a random error in gene replication that leads to a change

Describe the consequences of base substitutions, adding/removing nucleotides and transposons to protein expression

a substitution could: change a codon to one that encodes a different amino acid and cause a small change in the protein produced or even code for a stop codon. For example, sickle cell anemia is caused by a substitution in the beta-hemoglobin gene, which alters a single amino acid in the protein produced. Insertion or deletion of one or more nucleotides during replication can also lead to another type of mutation known as a frameshift mutation. The outcome of a frameshift mutation is complete alteration of the amino acid sequence of a protein. A transposable element (TE, transposon, or jumping gene) is a DNA sequence that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size. Transposition often results in duplication of the same genetic material.

polycistronic

a transcript that carries multiple genes (The protein encoded on a polycistronic message generally have related functions, allowing a cell to express related genes as one unit.)

- Suggested regimen: Active TB

all 4 drugs daily for 2 months then taken 3 times a week for an additional 4 months

genome

all of an organism's genetic material

Inducible

are not routinely produced at significant levels; instead, their synthesis can be turned on when needed. Helps prevent wasting material, often involved in the transport and breakdown of specific energy sources.

Repressible

are produced routinely, but their synthesis can be turned off when they are not required. The enzymes generally involved in biosynthetic pathways, such as those that produce amino acids. Cells require a sufficient amount of given amino acids to multiply; so, if an amino acid is not available in the environment, it needs to be produced by the cell. When amino acid is available, however, synthesis of the enzymes used in its production would waste energy

Constitutive

are synthesized constantly; the genes that encode these enzymes are always active. Usually play essential roles in the central metabolic pathways. For example, the enzymes of glycolysis are constitutive.

If transcription of a gene is turned "on", transcripts will

be available for translation

base substitution

changes the base of a single DNA nucleotide

DNA is ____-stranded and RNA is ____-stranded.

double/single (so RNA is shorter)

X-rays

ds and ss breaks in DNA can be lethal

intercalating agents

ethidium bromide inserts into ds DNA causing gaps, frameshift mutation occurs

Identify TB symptoms and complications such as XDR-TB

form of tuberculosis caused by bacteria that are resistant to some of the most effective anti-TB drugs. XDR-TB strains have arisen after the mismanagement of individuals with multidrug-resistant TB(MDR-TB). Symptoms of XDR-TB are no different from ordinary or drug-susceptible TB: a cough with thick, cloudy mucus (or sputum), sometimes with blood, for more than 2 weeks; fever, chills, and night sweats; fatigue and muscle weakness; weight loss; and in some cases shortness of breath and chest pain.

the functional unit of the genome is a

gene

point mutation

gene mutation in which a single base pair in DNA has been changed

TB Direct Observed Therapy Short-course (DOTS)

healthcare workers administer and watch patients as they take their medication

The lac operon contains three genes:

lacZ, lacY, and lacA. These genes are transcribed as a single mRNA, under control of one promoter. Genes in the lac operon specify proteins that help the cell utilize lactose. lacZ encodes an enzyme that splits lactose into monosaccharides (single-unit sugars) that can be fed into glycolysis. Similarly, lacY encodes a membrane-embedded transporter that helps bring lactose into the cell

UV irradiation

light energy causes covalent bonds to form between two thymine bases (thymine dimers), cell tries to repair with SOS system, BUT several base substitutions occur

Distinguish between mutation and horizontal transfer

most mutations are harmful to the bacterium. Horizontal gene transfer, on the other hand, enables bacteria to respond and adapt to their environment much more rapidly by acquiring large DNA sequences from another bacterium in a single transfer

knockout mutation

mutation that renders the protein product dysfunctional (spontaneous)

quorum sensing

the regulation of gene expression in response to fluctuations in cell-population density Who's around me? - Using two component system or uptake of cell signals to dictate bacterial group behavior

Central Dogma Theory

theory that states that, in cells, information only flows from DNA to RNA to proteins

horizontal gene transfer

transfer of genes between cells of the same generation

Recommended drugs for active TB:

· Isoniazid · Rifampin · Pyrazinamide · Ethambutol

Multidrug resistant strain of TB (MDR-TB):

· M. tuberculosis exhibits spontaneous chromosomal mutations · Mutations in RNA polymerase weaken binding affinity of rifampin, mutations alter binding site · pncA gene encodes pyrazinamide which converts pyrazinamide to its active form, pyrazinoic acid. Mutations in gene accounts for majority of resistant M. Tuberculosis

acid fast cell wall

· Nearly impermeable. · Resistant to drying, disinfectants, strong acids and bases · Special stain (acid fast stain) is required to identify bacteria of genus mycobacterium

characteristics of tuberculosis

· Obligate aerobe (gram +) · Acid fast · Non motile bacillus · Long generation time (>16 hours) · No endospore formation · Various metabolic pathways