Unit 2 - Learning Objectives

Why is the genome important for DNA storage?

It contains bases that are transcribed into RNA and translated into amino acid chains that make up the protein necessary for cellular structure, function, survival and reproduction

What can genetic sequencing tell us?

It can tell us about a microorganism’s characteristics such as identity, metabolic pathways, energy production, nutrition requirements, and phylogentic relationships without having to culture the cell

What can performing targeted sequencing allow us to do?

It allows us to compare one gene in many organisms and look for mutations with a gene in a population of organism

What type of genomic sequencing would be best to perform if you were looking to identify genetic differences between different biochemical pathways for the same process in different microorganisms

Targeted sequencing

What can performing whole genome sequencing allow us to do?

It allows us to study all genes in one or more organisms and to discover regulation, pathways, and metabolism in uncultured organisms

If you were trying to determine a characteristic of an organism, such as it’s metabolic pathways, which type of genetic sequencing would be best to use?

Whole genome sequencing

What are the features of an ORF (open reading frame)?

A ribosomal binding site followed by a start codon, which begins the coding sequence which is ended by the stop codon

How does the computer analyze DNA sequencing data to find the ORF?

A possible start codon is identified → a possible stop codon is identified → computer counts the number of bases between possible start and stop codons to ensure they are a multiple of 3 → identifies a possible ribosomal binding site → computer calculates codon bias in ORF → computer decides if ORF is likely to be genuine → computer adds it to the list of probable ORFs

Why can parasitic bacteria exist with smaller genomes than microbes that are free living?

Parasitic bacteria require fewer metabolic pathways than free living bacteria because they can synthesize more nutrients directly from the host

What can genome sequences tell us about uncultured bacteria?

Their identity, metabolic pathways, energy production, nutrient requirements, and phylogenetic relationships

Why can the vast majority of microbes only be studied using genetic sequencing?

99% of all prokaryotes cannot be cultured in the laboratory because they live in complex communities that cannot be replicated in cultures

What does branch length tell us about the evolutionary relationship between organisms?

It is proportional to the evolutionary distance of the organism at the end of the branch to the organism present at the node

What information does comparative genomics provide us when comparing two organisms?

It allows us to identify differences between two organism’s genomes which can help us determine the effect of different genes and how different organisms relate to each other

Which gene is usually used to determine evolutionary relatedness between organisms?

Small subunit ribosomal RNA gene

Why is small subunit ribosomal RNA gene usually used for determining evolutionary relation between genes?

This gene has the same functions within all organism and can be shorted to just looking at variable regions to reduce needed computing power as the other sections rarely change

Why is using genes with the same function important for determining evolutionary relation between species?

Genes that have different functions usually evolve in different ways

What are other genes besides small subunit rRNA genes that are used for phylogentic comparison?

Genes for ATPase and glycolysis enzymes

What are advantages of having a large genome?

There are lots of genes that allow survival in a variety of habitats

What is a disadvantage of having a large genome?

Carrying a large genome requires more resources than carrying a small genome

How are microorganism’s identified through nucleic acid hydribization?

DNA from two organism’s, one known and one unknown, is added together in solution and heated so that DNA strands separate before slowly being cooled to reform them - matching strands for different organism will hybridized when cooled and if >70% of DNA from each organism hybridizes, it indicates they are the same species

How are microorganism’s identified through Southern blotting?

Flurescently tagged DNA are used to identify an unknown species based on if that specific sequence of DNA is present

How are microorganism’s identified through DNA chips?

A chip contains many different fluorescently tagged DNA probes that bond to species specific DNA sections of an unknown pathogen to identify the species

How are microorganism’s identified through ribotyping?

Looks at the rRNA of the small ribosome subunit to determine the phylogeic relationship between organisms and to use the specific sequence to determine the species

How are microorganism’s identified through flurescent in site hybridization (FISH)?

Cells are treated to allow DNA probes to bind with DNA to determine the identify, abundance,and relative activity of microorganisms within an environment

What are the steps of genome sequencing?

Sequences are aligned into “contigs” → scaffolds → “gap closure” and “finishing” → gene prediction → annotation

What happens during the “gap closure” stage of genome sequencing?

Regions of low sequence data are identified

What happens during the “finishing” stage of genome sequencing?

The scaffolds are synthesized into a draft genome

What happens during the “gene prediction” stage of genome sequencing?

Computer predicts the location of genes

What happens during the “annotation” stage of genome sequencing?

Function of genes are assigned based on sequence similarities between the sequenced genome and the unknown genome

True or false: alignments can only be done for sequences read from a single microorganism, not for an entire microbial community containing hundreds of species

False

How is the DNA replication fork formed?

Topoisomerases unwind the supercoils of the DNA as helicase unwinds small sections to form the replication fork

How does DNA replication occur at the replication fork?

Moving 3’ to 5’, the leading strand is continuously synthesized moving away from the replication fork moves while the lagging strand is synthesized in short Okazaki fragments moving away from the replication fork which are restarted each time the fork moves and joined by DNA Ligase

Can DNA polymerase start synthesizing new DNA strands alone?

No, they require RNA primers to begin synthesizing daughter strands or Okazaki fragments

How does the nucleotide sequence of one DNA strand determine the sequence of the complimentary DNA strand?

Bases only bind to one other specific base: adenine binds to thymine and guanine binds to cytosine so based on the sequence of one DNA strand the other strand must be ordered in such a way that each base always pairs with it’s complimentary base

How does the nucleotide sequence of one DNA strand determine the sequence of the transcribed mRNA strand?

Each base on DNA can only pair with one base on the mRNA strand: adenine on the DNA strand binds with uracil on the mRNA strand, guanine binds with cytosine and thymine on the DNA strand binds with adenine on the mRNA strand so the mRNA strand must be synthesized in such an order that each base is paired with it’s complimentary base

What does it mean that two strands of DNA are complementary?

Each base only pairs with one other specific base such that adenine pairs with thymine and guanine pairs with cytosine

Why do cells perform metabolic regulation?

To prevent the allocation of energy and materials to developing unneeded products through transcription of DNA to mRNA and translation to proteins

Describe rho-dependent termination

It relies on the presence of a rho protein to unwind DNA after the end of the coding region causing DNA to unbind

Describe intrinsic termination

It occurs independently of a protein by the bases in the synthesized mRNA strand after the structural genes binding to form a stem and loop that causes RNA polymerase to unbind from the DNA

Dark blue

Regulatory genes

Orange

Promotor

Yellow

Operator

Green

Structural genes

What sequence comes before a promotor in an operon and controls mRNA synthesis?

Activator-binding site

How do regulon’s function to control gene expression of multiple genes?

Related operons are all controlled by the same regulator gene or proteins which allows multiple operons with related genes to be inhibited or activated by one molecule or protein

True or false: operons can only contain one structural gene

False

True or false: a new strand of mRNA is synthesized for each structural gene of an operon

False

How many proteins are produced from an operon?

A number of proteins equal to the number of protein coding structural genes an operon contains

What are the two genetic elements that compose the genome in prokaryotes?

Chromosomes and plasmids

What is the advantage of coupling transcription and translation for metabolic adjustment?

Prokaryotic cells can quickly generate new metabolic pathways to adapt to new environments and nutrient availability

Why will one mRNA transcribed from an operon have muliple ribosomes bound to it?

Operons contain more than one structural gene and each gene contains an individual ribosomal binding site so multiple ribosomes can bind at a time to translate each gene simultaneously

What are the advantages related to metabolic regulation for organizing genes into operons?

Genes for one metabolic pathway can all be expressed at once and controlled by one or two regulatory proteins which prevents incomplete pathway synthesis

What is the benefit of gene expression being influenced by external molecules?

It allows cells to adapt to their environment

What are the benefit of gene expression being influenced by internal/cellular molecules?

It controls the production of molecules within a cell to prevent unnecessary use of energy and materials

Explain the transcription regulation of the arginine in a solution without arginine

When no arginine is present, the repressor cannot bind to the operon, so RNA polymerase can proceed in transcribing the structural genes

Explain the transcription regulation of the arginine in a solution with arginine

When arginine is present, it acts as a corepressor, creating a change in the structure of the repressor so that it binds to the arg Operator, preventing RNA polymerase from transcribing the structural genes

Explain the transcription regulation when lactose and glucose is present

Allolactose is present and acts as an inducer, changing the shape of the repressor causing it to unbind from the lac operator but glucose levels are high which means cyclic AMP is low so CRP protein cannot bind to the activator-binding site so RNA polymerase cannot bind to the lac promotor and transcription cannot proceed to proceed

Explain the transcription regulation when lactose and glucose are both not present

With glucose not present, cyclic AMP concentration is high and it acts as an inducer to the CRP protein allowing it to bind to the activator-binding site, causing RNA polymerase to bind to the lac promotor but since lactose is not present, the repressor is still bound to the lac operator preventing transcription from proceeding

Explain the transcription regulation when lactose is present but glucose is not present

With glucose not present, cyclic AMP concentration is high and it acts as an inducer to the CRP protein, allowing it to bind to the activator-binding site, causing RNA polymerase to bind to the lac promotor. Since lactose is present allolactose acts as an inducer to the repressor, causing it to unbind from the lac operator and transcription to proceed

Explain the transcription regulation when glucose is present and lactose is not present

With glucose levels high, cyclic AMP concentrations are low so CRP protein cannot bind to the activator-binding site and RNA polymerase cannot bind to the lac promotor. Since lactose is not present, the repressor is still bound to the lac operator which would prevent RNA polymerase from transcribing the structural genes even if it could bind so transcription does not proceed

Explain transcription regulation of the maltose operon when maltose is present in the medium

Maltose acts as an inducer to the maltose activator protein, allowing it to bind to the activator-binding site which allows RNA polymerase to bind to the mal promotor and transcription to proceed

Explain transcription regulation of the maltose operon when maltose is not present in the medium

The maltose activator protein cannot bind to the activator-bind site which prevents RNA polymerase from binding to the mal promotor and transcription from proceeding

How does transcription regulation of the tryptophan operon differ from the lactose, arginine, and maltose operon?

It’s controlled by feedback inhibition so that when excess tryptophan is present, it acts as a corepressor which allows a repressor to bind to the tryptophan operator and prevent further tryptophan synthesis

How does the lactose operon differ from the tryptophan, arginine and maltose operons?

It needs both a activator protein (CRP) and an inducer to bind to the repressor molecule to allow for transcription

Explain why anabolic pathways are typically transcriptionally regulated by negative or repressive controls

Since these pathways are only useful if the molecule being synthesized is not present, that molecule can act as a corepressor and allow a repressor to bind to the operon when the molecule is present, preventing unnecessary transcription

Explain how anabolic pathways are transcriptionally regulated by negative or repressive control by using arginine

When arginine is present in the environment, the cell does not need devote energy to producing it so it acts as a corepressor and allows a repressor to bind to the arg operator preventing transcription but when it is not present, it is necessary for the cell to synthesize so the repressor cannot bind to the operator allowing transcription to occur

Explain why catabolic pathways are typically transcriptionally regulated by positive controls (inducers)

Since these pathways are only useful when the molecule is present to be broken down, by the molecule functioning as an inducer it can either bind to repressors causing them to unbind from the operon or bind to activator proteins, allowing them to bind to the activator-binding site and RNA polymerase to bind the promotor which allows transcription to proceed

Explain how catabolic pathways are transcriptionally regulated by positive controls (inducers) using maltose

Proteins to catabolize maltose are not useful unless maltose is present to be used as a fuel source so when maltose is present it acts as an inducer, allowing the maltose activator protein to bind to the activator-binding site and allowing RNA polymerase to bind to the mal promotor and transcription to proceed

How is the transcription negative control in the arginine and lactose operon different?

In the lactose operon, allolactose which is present when lactose is present acts as an inducer causing the repressor protein to unbind from the operator and RNA polymerase to proceed if it is bound while in the arginine operon, arginine when present prevents transcription by acting as a corepressor

How is the transcription negative control in the arginine and lactose operon similar?

In both, repressors prevents unnecessary transcription

Out of the arginine, tryptophan, lactose and maltose operator, which uses inducers and how?

In both maltose and lactose operons, both substrates act as in inducers, maltose binds to the maltose activating protein allowing RNA polymerase to bind and transcription to proceed and in the lactose operon, allolactose acts as an inducer to the repressor protein causing it to unbind from the operator, both of which allow transcription to proceed but cyclic AMP also acts as an inducer to CRP which allows it to bind to the activator-binding site which allows RNA polymerase to bind

Out of the arginine, tryptophan, lactose and maltose operator, which uses corepressors and how?

In both arginine and tryptophan, both molecules act as corepressors and prevent transcription by allowing repressors to bind to the operators but in arginine operons it’s extracellular arginine while in typtophan operons it’s intracellular trytophan through feedback inhibition

Why do cells grown in both glucose and lactose exhibit diauxic growth?

Originally, only glucose is used due to catabolite repressor, allowing the cell to grow at it’s maximum rate but once glucose is exhausted, cyclic AMP concentration increases which allows the activator protein to bind to the activator-binding site and lactose already has already unbound the repressor protein so the transcription proceeds to synthesize a new metabolic pathway for catabolism of lactose but enzyme synthesis takes time, resulting in the lag where no population growth is observed and then growth with lactose is slower than that with glucose so a different growth rate is observed

Why do cells use post-transcriptional regulation of metabolic pathways?

It provides a way for cells to control metabolic activity once enzymes are translated from mRNA strands and prevent translation of mRNA after transcription

What do riboswitches and small RNA molecules (sRNA) have in common and how do they differ?

Both regulate translation of a mRNA strand, controlling protein synthesis but in sRNA, mRNA strands are destroyed while with riboswitches, the presence of some molecule prevents transcription by blocking ribosomal binding site

How do riboswitches and small RNA molecules (sRNA) differ from feedback inhibition?

Feedback inhibition prevents enzymes from functioning to control metabolic pathways while riboswitches and sRNA prevent translation of mRNA

What is another name for feedback inhibition?

Allosteric regulation of enzymes

How are riboswitches and feedback inhibition similar?

Both occur due to the presence of a small molecule and neither destroy the mRNA strand or protein that is effected so that it can be used again later

What is positive or direct selection of mutants?

A method of mutant detection that rejects nonmuntant cells, usually used for identifying resistant strains

How can mutants be directly selected for?

Usually done by culturing cells in a medium that contains a compound, like an antibiotic, that would prevent wildtype cells from growing and only allow cells with a mutation to provide resistance to that compound to grow

How can mutants be identified through negative or indirect selection?

Uses replica plating where cells are cultured on a master plate and a surface is used to transfer colonies onto one plate with an identical medium and another plate with a medium lacking a some compound that the wildtype cells are known to be able to synthesize - cells with colonies on the first but not the second plate can be identified as mutants

How can mutations impact a cell’s growth or survival in a specific environment?

Mutations can be disadvantageous if they reduce the function of or inactivate proteins necessary to cellular structure or metabolic pathways needed for the environment or can be beneficial and allow increased growth in an environment if they enhance a protein’s function or produce a product that better utilizes the environments resources

Why are mutation important to cell survival in challenging environments?

Mutation can create variation that can occasionally improve protein function or create new proteins that better allow cells to survive, allowing populations to survive in a range of environments - if the mutation allows increased survival in an environment, mutants will replicate more rapidly and persist at a higher rate in that population

What kinds of mutations would lead to a neutral change?

Silent mutations and missense mutations that result in an amino acid being produced by the altered codon with similar characteristics that is encoded for in the wildtype codon

What kinds of mutation almost always lead to nonfunctional proteins?

Nonsense mutations

Which mutations lead to large changes to the amino acids of, and therefore the characteristics of, an encoded protein?

Frameshift mutations

What is this mutation: ATG GAA CTC ACC → ATG AAC TCA CC…

Frameshift deletion

What is this mutation: ATG GAA CTC ACC → ATG GCA ACT CAC…

Frameshift insertion

What is this mutation: ATG GAA CTC ACC → ATG GAA UAG (stop codon) ACC

Nonsense mutation

What is this mutation: ATG GAA CTC ACC → ATG GAA UAG CCC

Missense mutation

What effect does a missense mutation have on the function of a protein?

It may have a large effect, usually reducing function, if the amino acid encoded for after the mutation has different characteristics than the original amino acid encoded for or it may have no effect if the new amino acid and the original amino acid have similar characteristics

What effect does a nonsense mutation have on the function of a protein?

Likely renders it inactive

What effect does a silent mutation have on the function of a protein?

It has no effect

What effect does a frameshift mutation have on the function of a protein?

It likely has a large effect on the proteins function, usually reducing it’s function or changing its function entirely

What effect does a reversion mutation have on the function of a protein?

It returns the protein’s function to something similar to it’s original prior to the mutation, usually returns protein function

During a mutation, what molecule actually changes?

Nucleotides

Based on the central dogma of molecular biology, explain a base pair substitution mutation?

One or more bases in the structural gene section of the open reading frame are changed to a different base, which is translated as a different base in the mRNA strand which may change the amino acid the codon(s) with the changed base(s) codes for when translated by the ribosome which can change the structure and function of the encoded protein

What mutations are included in base pair substitutions?

Silent, missense and nonsense

Based on the central dogma of molecular biology, explain a frameshift mutation?

1+ nucleotide bases in DNA are either inserted or deleted into the structural gene section of the open reading frame which results in a change to translational reading frame of the transcribed RNA when codons are grouped to be translated as ribosome resulting in almost all codons changing, changing the structure and function of the protein