MBIO Lecture Exam 3

What different parts of the microbial cell are affected by microbial control agents?

- Alteration of membrane permeability - Damage to proteins (enzymes) - Damage to nucleic acids

What are the parameters that determine the effectiveness of an antimicrobial treatment?

- Number of microbes - Environment (organic matter, temperature, and biofilms) - Time of exposure - Microbial characteristics

Why is a primer required for DNA replication? Would the primer be found on the lagging strand, the leading strand or both? If it is found on both strands, how does the number of primers differ for each strand?

- RNA primer - Lagging strand

What are the "restrictions" of the DNA polymerase III enzyme?

- Requires a primer, because it must have a pre-existing 3' OH group in which to add a nucleotide. - Requires a template strand of DNA, it cannot simply start synthesizing a random strand of DNA> - It ALWAYS synthesizes DNA in the 5' à3' direction.

What are the different types of RNA? Which of these RNA molecules will be translated into a protein?

- Ribosomal RNA (rRNA) - Transfer RNA (tRNA) - Messenger RNA (mRNA) - "Other" (siRNA, miRNA, snRNA) ** Messenger RNA (mRNA)

Given a blank template showing a double-stranded DNA molecule undergoing replication, be able to identify which strand would serve as the leading strand and which would be the lagging strand.

- The polymerase working on the opposite strand will be moving away from the replication fork. - This is referred to as the lagging strand. - Because the polymerase synthesizes DNA in only 1 direction (5' à3'), one of the polymerases will be moving in the same direction as the replication fork. - We refer to this strand of DNA as the leading strand.

After termination the two circular chromosomes that result from replication are interlocked or __________________________ and must be separated by an enzyme called _________________________________ .

- concatenated - Topoisomerase IV

What are the three different ways that bacteria may take in foreign DNA? Compare and contrast these methods.

1. Transformation- The nonspecific uptake of foreign DNA from the environment- Does not require cell-to-cell contact- Integrates the foreign DNA its own chromosome 2. Conjugation- Requires direct cell-to-cell contact- Occurs between specific types of cells- F+ cells and F- cells 3. Transduction- Bacterial DNA is transferred from a donor cell to a recipient cell through a virus- It infects the bacteria

In a circular prokaryotic chromosome, how many replication forks would be present?

2

What is the end result of DNA replication?

2 identical double-stranded DNA molecules

What is the template for the DNA polymerase III?

3' OH group to a 5' phosphate group

In a circular prokaryotic chromosome, how many DNA polymerase III molecules would be present?

4

In what direction is an RNA molecule synthesized?

5' -> 3'

What are the possible outcomes of a mutation?

A mutation in a DNA sequence can result in changes in protein sequence. • Change in protein structure • Point mutations • Silent • Missense • Nonsense • Insertion or Deletion- Frameshift Harmful Neutral Beneficial

How does a repressible operon differ from an inducible operon?

An inducible operon requires an inducer molecule to stimulate transcription either by inactivating a repressor protein in a negative inducible operon or by stimulating the activator protein in a positive inducible operon. In a repressible operon, transcription is turned off either by the repressor becoming active in a negative repressible operon or by the activator becoming inactive in a positive repressible operon.

Which treatments are effective against endospores?

Autoclaves

Single-stranded Binding Protein

Bind to single-stranded DNA to prevent hydrogen bonding between DNA strands, reforming double-stranded DNA

What is a biocide? Algicide? Fungicide?

Biocide (germicide) - treatment that kills microbes (usually with certain exceptions, such as endospores) - chemical agent that inactivates microorganisms - Biocides generally have a broader spectrum of activity than antibiotics• - Biocides generally have a wider array of targets. Fungicide - kills fungi Algicide - kills algi

Commercial sterilization is a limited heat treatment meant to destroy what organism specifically?

C. botulinum

What does it mean if a gene is constitutively expressed?

Constitutively expressed genes, that is, are always "on" Many genes are expressed constituitively which means they are constantly being expressed. • Housekeeping genes

What is disinfection? Antisepsis? What is the difference between disinfection and antisepsis?

Disinfection is destroying harmful microorganisms Antisepsis is destroying harmful microorganisms from living tissueDisinfection usually refers to the destruction of vegetative (non-endospore forming) pathogens Antisepsis is used on living tissue while disinfectant is used for like tables and tabletops and what not

In a circular prokaryotic chromosome, where would replication begin?

Double-stranded DNA molecule Initiation

What is translation?

During the process of translation the cell is changing languages. It is taking a RNA template (nucleotides), and using it to build a polypeptide (amino acids).

What molecule is translated, and what is it translated into?

During the process of translation the cell is changing languages. It is taking a RNA template (nucleotides), and using it to build a polypeptide (amino acids). RNA into a protein

What are some key differences between replication in prokaryotes and eukaryotes?

Eukaryotic Replication • Requires the replication of multiple, linear chromosomes. • The human genome has been found to have thousands of origins of replications. • Replication is a slower process (100 bp/sec). Prokaryotic chromosomes generally have one origin of replication

What is bacteriostasis?

Inhibiting, not killing, microbes

Topoisomerase IV

Introduces sing-stranded break into concatenated chromosomes to release them from each other, and then reseals the DNA

Why is the telomere composed of noncoding DNA?

It is an evolutionary benefit because of the end replication problem. The enzyme telomerase's job is to extend the original parental DNA using its own template. It protects chromosomes from damage.

Understand that in prokaryotes, transcription and translation happen simultaneously. Why is this not possible in eukaryotes?

It is not possible in Eukaryotes because those are physically separated by the nucleus.

What does it mean if something is bacteriostatic? What is the difference between bacteriocidal and bacteriostatic?

It means to stop or steady or inhibit the growth and multiplication of bacteria Bacteriocidal kills bacteria while bacteriostatic just inhibits or stops the bacteria from growing

What is sanitization? Is this the same as sterilization?

Lowering microbial counts on eating utensils to safe levels No sterilization is the removal of all living microorganisms

DNA Polymerase III

Main enzyme that adds nucleotides in the 5' to 3' direction

What is decimal reduction time?

Minutes to kill 90% of a population at a given time

How does moist heat sterilization act on microorganisms? What are the examples of moist heat sterilization?

Moist heat sterilization denatures proteins. - Boiling- Free-flowing steam

Is pasteurization a method of sterilization?

No

Is boiling an effective method of sterilization?

No it is not. Endospores and viruses are not always destroyed this quickly and can be boiling for more than 20 hours

Helicase

Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases

What is pasteurization? Why is it a treatment used for dairy products (why don't we use an autoclave instead)? Understand that in pasteurization if you decrease the temperature of exposure then you may need to increase the length of time the item is exposed to that temperature. What is High-temperature short-time?

Pasteurization eliminates pathogenic microbes. It also lowers microbial numbers, which prolongs milk's good quality under refrigeration HTST - most milk pasteurization today uses temperatures of at least 72 degrees Celsius but only for 15 seconds

How is gene expression affected by environmental conditions, does this affect the genotype or the phenotype?

Phenotype is determined by the specific genes within a genotype that are expressed under specific conditions. Although multiple cells may have the same genotype, they may exhibit a wide range of phenotypes resulting from differences in patterns of gene expression in response to different environmental conditions.

What are the different types of mutations? What happens to the polypeptide in each case?

Point mutation (base substitution) - a single base at one point in the DNA sequence is replaced with a different base. When the DNA replicates. the result is a substituted GG, or CG for GC.Missense mutation - when an incorrect base causes the insertion of an incorrect amino acid in the protein. Nonsense mutation - when there is a nonsense (stop) codon in the middle of an mRNA molecule that creates only a fragment of the protein being synthesized Frameshift mutation - when one or a few nucleotide pairs are deleted or inserted in the DNA

What enzyme is responsible for the process of transcription?

RNA polymerase

Where does translation occur?

Ribosome

What cellular components are necessary for translation?

Ribosome (rRNAand proteins) RNA template tRNA Amino acids Translation Factors

Ligase

Seals the gaps between the Okazaki fragments on the lagging strand to create one continuous DNA strand

What is sepsis? Asepsis?

Sepsis refers to bacterial contaminationAsepsis is the absence of significant contamination- aseptic surgery techniques prevent the microbial contamination of wounds

Where does translation start and stop?

Start- AUG Stop- nonsense codons

What is sterilization? What is the difference between sterilization and commercial sterilization?

Sterilization is removing and destroying all microbial life Commercial sterilization is killing C. botulinum endospores from canned goods

Primase

Synthesizes RNA primers needed to start replication

In a circular prokaryotic chromosome, where would replication end?

Termination

What happens in the A, P and E site of the ribosome?

The A site accepts an incoming tRNA bound to an amino acid. The P site holds a tRNA that carries a growing polypeptide (the first amino acid added is methionine (Met)). The E site is where a tRNA goes after it is empty, meaning that it has transferred its polypeptide to another tRNA (which now occupies the P site).

What is the central dogma of molecular biology?

The central dogma essentially is investigating the processes in which this information is converted from the form which we find within DNA to the functional form, which are often proteins or functional RNA. 1. Replication - start out with double stranded DNA and end up with double stranded DNA. 2. Transcription - use DNA as a template to build RNA. 3. Translation - RNA is used as a template to build proteins.

What is a telomere?

The end segments of chromosomes that contain an abundance of noncoding DNA

What is a telomerase, and how does it add DNA without a template?

The end of chromosomes that contain an abundance of non-coding DNA. Require special telomerase for replication.

Microbial death is exponential, how does this relate to the idea that total microbial numbers affects the amount of time a surface should be exposed to a disinfectant?

The higher the microbial number, the longer the surface should be exposed to the disinfectant.

Understand the examples given for regulation of gene expression. How does expression change in the presence/absence of an inducer? What is the role of a repressor? Where does it bind? How does an inducer interact with a repressor?

The inducer initiates transcription. The repressor protein binds with the operator and prevents transcription from the operon. The inducer then binds to the repressor protein and the inactivated repressor can no longer inhibit transcription. The structural genes are transcribed, ultimately resulting in the production of the enzymes needed for catabolism (lactose catabolism?)

What are Okazaki fragments, and on which strand (leading or lagging) would they be found?

The lagging strand is synthesized in fragments, referred to as Okazaki fragments.

How are mRNA, tRNA and rRNA all involved in translation?

The mRNA (messenger RNA) carries the info regarding what protein is to be made. ... The tRNA (transport RNA) carries the amino acid to the rRNA. The rRNA (ribosomal RNA) makes up the ribosome. The ribosome builds the protein according to the instructions written in the mRNA with the amino acids ferried in by the tRNA.

What is degerming? What is an example of degerming?

The mechanical removal of microbes from a limited area Swabbing skin with alcohol before an injection

What does it mean that DNA replication is semiconservative?

The original DNA molecule is separated, each strand serves as a template for the synthesis of a new, complementary strand which remains annealed to the "old" template strand. This results in 2 molecules of DNA, each containing one "old" strand (in blue) and one "new" strand (in red).

How does the steam in an autoclave reach such high temperatures?

The pressure causes the temperature to increase

DNA Replication

The process by which a doublestranded DNA molecule is separated and each strand acts as a template for synthesis of a complementary strand of DNA. The result of replication is 2 identical double-stranded DNA molecules. This process is semiconservative.

What is a promoter composed of, and what is the function of a promoter?

The promoter is a specific sequence of nucleotides found on the DNA template. The function of the promoter is to mark the presence of a gene on the DNA.

What does it mean to regulate gene expression? Why is it important that cells be able to regulate gene expression? Are all genes regulated in the same way?

The regulation of gene expression conserves energy and space. It would require a significant amount of energy for an organism to express every gene at all times, so it is more energy efficient to turn on the genes only when they are required.

What is vertical gene transfer? What is horizontal gene transfer?

The transfer of genes from parents to offspring Horizontal gene transfer is getting your DNA from something other than your parents

What is the universal code? And what is the difference between sense and nonsense codons? What is the start codon, and what does it encode for?

The universal genetic code is made up of several codons or triplet bases. ... Due to this, of the 64 codons, 61 codons code for the 20 amino acids. There are two punctuation marks in the genetic code called the START and STOP codons which signal the end of protein synthesis in all organisms. •Sense codons - 61 of the codons encode for a specific amino acid •Nonsense codons "stop codons" - 3 of the 64 possible codons do not encode for an amino acid.

Where does transcription start and stop?

Transcription begins when the RNA polymerase binds to the DNA at a site called the promoter. Only one of the two DNA strands serves as the template for RNA synthesis for a given gene. RNA synthesis continues until RNA polymerase reaches a site on the DNA called the terminator

What is transcription?

Transcription is when a DNA template is used to synthesize a complementary strand of RNA (RNA transcript)

Transcription, Translation, Replication: Location

Transcription: Translation: Replication:

Transcription, Translation, Replication: Other Involved Proteins

Transcription: Translation: Replication:

Transcription, Translation, Replication: Synthesizing Machinery

Transcription: Translation: Replication:

Transcription, Translation, Replication: Stop signal/site

Transcription: Translation: Stop codon Replication:

Transcription, Translation, Replication: Start signal/site

Transcription: +1 site Translation: start codon Replication:Origin of replication

Transcription, Translation, Replication: Product

Transcription: A strand of RNA Translation: Polypeptide Replication: DNA

Transcription, Translation, Replication: Template

Transcription: DNA Translation: tRNA Replication: mRNAtr

When DNA is denatured what does this mean?

Two strands of the DNA helix are separated The process of breaking a double-strand of DNA into single strands

Is DNA polymerase III capable of proofreading its own work?

YES It can "proofread" its own work, considerably decreasing the number of errors that occur in the newly synthesized strand.

Can a disinfectant be used as an antiseptic?

Yes but some chemicals might be too harsh to use on living tissue

Monod once famously said "What is true in E.coli is true in an elephant". What is the significance of this statement?

You are the results of the proteins that your cell makes. We can study E. coli and learn how other organisms work.

What is the template for transcription?

antisense strand

What are the codon and the anticodon?

codon- a sequence of three nucleotides on a mRNA molecule that encode a specific amino acid. anticodon - a sequence of three nucleotides on a tRNA molecule that bond to a complementary sequence on an mRNA molecule. The anticodon sequence determines the amino acid that the tRNA carries.

Are all genes regulated?

no, not all some genes are not regulated at all

What is a mutation? How do mutations play into the theory of natural selection?

• Changes in a cell's DNA introduces genetic variation into the population. • Sometimes these changes can make that organism more physically fit for the current environment. • Natural selection • Mutations are the permanent change in the base sequence of DNA • A mutation in a DNA sequence can result in changes in protein sequence. • Change in protein structure • Point mutations • Silent • Missense • Nonsense

What is the function of DNA?

• DNA carries the instructions of the cell.

DNA Polymerase I

• DNA polymerase I is responsible for removing the RNA primers, and replacing the RNA with DNA resulting in a final molecule that is made entirely of DNA.

Initiation of Replication

• Does NOT begin at a random location • Origin of replication- oriC • Prokaryotic chromosomes generally have one origin of replication • Eukaryotic chromosomes may have multiple origins Origin In order to synthesize new DNA strands, the "old" double-stranded DNA must be separated.

Why is it important that DNA be replicated with a high degree of accuracy?

• During cell division a copy of these instructions must be made with the highest possible degree of accuracy. It has to be close to perfect. That is all the new cell is getting, the copy that we made. If it is not perfect or close to perfect then that cell may not be able to survive.

What does the term bidirectional replication mean?

• Helicase movement results in the formation of a replication fork. • Two helicases will work in opposite directions at the oriC resulting in two replications forks. • Bidirectional Replication • Each fork will result in the replication of approximately half of the chromosome, meeting at the termination site.

Understand the terminology of gene expression.

• Not all genes are regulated Gene expression is the process by which the information encoded in a gene is used to direct the assembly of a protein molecule. The cell reads the sequence of the gene in groups of three bases. Each group of three bases (codon) corresponds to one of 20 different amino acids used to build the protein.

How are plasmids replicated, and how does this differ from the replication of the chromosome?

• Plasmids are replicated using a method referred to as "Rolling Circle Replication". • The double-stranded plasmid is nicked, and DNA polymerase III begins synthesis of the new strand at the nick, simultaneously displacing the previous strand.

Telomerase

• Telomeres, the end segments of chromosomes that contain an abundance of noncoding DNA. • Require special enzymes called telomerases for replication.

Termination

• Termination of replication takes place approximately half of the length of the chromosome from the origin of replication. • There are specific sequences associated with termination (ter sequences). • After termination the two circular chromosomes are interlocked, or concatenated. • They must be separated so that the chromosomes can go to separate cells, this is done through the action of Topoisomerase IV.

Elongation

• The elongation stage is when the new complementary strands of DNA are being synthesized. • This occurs at a rate of approximately 1000 bp/sec. • The synthesis of the new strand is performed by an enzyme called DNA polymerase III.

What is the product of transcription?

• The end product of transcription is a strand of RNA

What are the three stages of DNA replication? Explain what happens at each stage.

• Three stages of replication 1. Initiation 2. Elongation 3. Termination

How does transcription differ in prokaryotes vs. eukaryotes?

• While the process of transcription is very similar in prokaryotes and eukaryotes there are some fundamental differences - Transcription in eukaryotes occurs in the nucleus. - The resulting transcript may contain both introns and exons. - The resulting transcript undergoes many processing steps prior to translation.