MIC 205: Chapter 7 - Microbial genetics (transcription, translation,

What is transcription?

Copying DNA information within a gene into RNA nucleotide sequences (DNA → RNA)

Where does transcription occur

In the nucleoid of prokaryotes & in the nucleus/mitochondria/chloroplasts of eukaryotes

What are the three types of RNA transcript

Messenger RNA (mRNA)
Transfer RNA (tRNA)
Ribosomal RNA (rRNA)

What is mRNA

Messenger RNA: temporary carrier of genetic information from chromosomes to ribosomes; translated for protein synthesis
(mRNA contains the code for the new polypeptide)

What is rRNA

Ribosomal RNA: form complexes with ribosomal proteins to form ribosomes that synthesize polypeptides
(rRNA combines with proteins to form ribosomes, which are the platform for protein synthesis)

What is tRNA

Transfer RNA: transport/transfer amino acids to ribosomes

What is used to carry out transcription

RNA polymerase in 3 steps

What is the start signal for transcription and what is it

Promoter: binding site on DNA for RNA polymerase

What aids RNA polymerase to locate the promoter in transcription

Sigma factor us used to help RNA polymerase locate the promoter

What is the stop signal for transcription

Transcription terminator

What is the coding sequence for transcription

The area in-between the promoter to the terminator

What are the 3 steps for transcription

Initiation, Elongation, and Termination

What in initiation in transcription

RNA polymerase recognizes and binds to promoter sequence with the aide of sigma on DNA template
(RNA polymerase with the sigma identifies the promoter and binds to it. Then RNA polymerase causes dehybridization of the 2 strands of the promoter to open and RNA polymerase can read 1 of the strands)

What is elongation in transcription

RNA polymerase extends the newly formed transcript using triphosphate ribonucleotides (5’ → 3’). It occurs in a complementary nature to the DNA strand
(RNA polymerase is moving down the templte, reading it, and at the same time, synthesizing a RNA molecule. Sigma factor is released) This is all anabolism as you take small subunits and link them together

What is termination in transcription

RNA polymerase detaches and releases transcript upon reaching terminator sequence on DNA template. This can occur in an rho-dependent or rho-independent nature
(once we reach terminator, it is telling us transcription is going to end)

What are 2 ways termination can happen in transcription

Rho-dependent: means polymerase gets to terminator, rho protein comes in and causes disassociation (meaning RNA polymerase comes off and RNA transcription is released)
Rho-independent
Rho is a protein (just need to know this is 1 of 2 ways you will terminate the process once you get to the end of transcription)

What is the difference in forming the gene by transcription versus DNA replication and where each starts

Transcription forms RNA molecules from genes
Replication forms DNA molecules from chromosomes and plasmids

Transcription starts at promoters (thousands/chromosomes)
Replication start at origin of replication (one/chromosome)

What is the difference between DNA polymerase versus RNA polymerase

DNA polymerase III will read and synthesize a new strand of DNA 5’ → 3’
DNA polymerase I will remove RNA primer and read DNA and synthesize a new strand of DNA 5’ to 3’
RNA polymerase can unwind and rewind template DNA on its own and synthesizes RNA transcripts 5’ → 3’ direction

What is translation

The synthesis of polypeptides (proteins) through interpretation of mRNA nucleotide sequences

Where does translation occur and what are the 3 steps in translation

Carried out in the cytosol in three steps → initiation, elongation, termination

What does the messenger RNA contain (mRNA) and what it is in translation

Codons: clusters of 3 nucleotides units in an mRNA molecule that codes for specific amino acid
mrNA encoded within it is the information on how to build proteins (what the order of amino acids is)

What are the special amino acids in mRNA translation

Start codon: AUG - not every AUG is a start codon, only those associated with the ribosomal binding site (ribosomal binding site RBS is towards the 5’ end of the mRNA molecule and start codons are towards the 3’ end/right of RBS so start codon is about 10 units away from RBS)
Stop codons: UAA, UAG, UGA - found at end to mRNA molecules to tell ribosomes to halt translation

What are the 2 parts of the transfer RNA in translation

Acceptor stem and anticodon

What is an acceptor stem in tRNA of translation

A portion of tRNA that is linked to an amino acid by aminoacyl-tRNA synthetase enzyme
The accepting stem is the 3’ end where specific amino acids is going to be linked to specific tRNA

What is anticondon in tRNA for translation

Nucleotide triplet complementary to the mRNA codon for a given tRNA’s amino acid

What does each tRNA carry in regards to mRNA

Each tRNA carries a specific amino acid and recognizes an mRNA codon only for that amino acid

How are prokaryotic 70s ribosome compared to eukaryotic 80s ribosome

Prokaryotic 70s ribosome are smaller than eukaryotic 80s ribosomes

What is initiation in translation

Ribosomal subunits assemble on mRNA transcript at the ribosomal binding site (RBS). Recruit initiator tRNA molecule

What is elongation in translation

Ribosome recruits amino acid-bound tRNAs according to the sequence of mRNA template. The ribosome will move 3 nucleotides
(you link what you get from the codons together by a peptide bond. this step is building the chain of proteins and it does not stop until stop codon)

What is termination in translation

Special release factors recognize stop codons, prompt ribosomes to sever polypeptide from last tRNA.
Ribosome then dissociates into its complement subunits and falls away from mRNA template
(there is no tRNA for the 3 stop codon. When we reach the stop codon, since there isn’t tRNA that binds, the ribosomes just pauses. Special release factors with enter into A sites and cause everything to disassociate and polypeptide gets free.)

How is transcription and translation coupled

Transcription doesn’t have to finish before translation and more transcription begins which is called coupled transcription translation. this happens because there is no compartmentalization in prokaryotic cells (which is why bacterial cells grow so fast)

What is a polyribosome

Multiple ribosomes translating a single mRNA molecule

What are house-keeping genes

¾ of genes that are expressed at all time (house-keeping genes are genes that are turned on all the time)

What are the other ¼ genes and why

Other genes that are regulated so they are only transcribed and translated when a cell needs them. This allows cells to conserve energy and cytoplasm isn’t crowed with unused enzymes

How do cells that want to stop making a protein do that

They typically shut down transcription of the corresponding gene. If necessary, a cell may halt all translation directly.
(with this we are talking about allowing a gene to turn on/off)

How are genes with similar function grouped/organized and what is it

Operons: group of bacterial genes that share 1 promoter only in prokaryotic)

What does a polycistronic mRNA carry

The information of several genes, which are translated into several proteins (meaning multiple messages in 1 mRNA molecule)

What are prokaryotic operons

Clusters of genes on a chromosome whose expression is regulated by a common promoter and operator elements

What are the 2 types of prokaryotic operons

Inducible operons and repressible operons

What is inducible operons

Must be activated by inducers like lactose (lac) operon

What are repressible operons

Transcribed continually until deactivated by repressors like tryptophan (trp) operon

What happens in an inducible lactose (lac) operon with no lactose present

These genes are involved in lactose catabolism (taking lactose and breaking it down and extracting energy).
If there is no lactose present in the environment, it (the operon) turns off and is repressed since repressor blocks operator
The regulatory gene undergo transcription and translation and we make our regulatory protein known as the repressor (which is specific for lactose operon). Now, the repressor binds to the operator and RNA polymerase with the sigma will bring the polymerase to the promoter for the operon but is blocked by the repressor.

What happens in an inducible lactose (lac) operon with lactose present

These genes are involved in lactose catabolism (taking lactose and breaking it down and extracting energy).
The regulatory gene does the same thing and mRNA makes the regulatory protein. The repressor wants to bind to the operator but since lactose is present within the cell, the lactose binds to the repressor and inactivates it, causing the repressor to not bind to the operator. Now the area is open and RNA polymerase with sigma cause bind to promoter and move its way through operator and get to structural genes, allowing transcription and translation to occur.

What happens in a repressible tryptophan (trp) operon with no tryptophan present

Tryptophan operon is a biosynthetic pathway for building amino acid tryptophan. It is an anabolic pathway, enzymes will be produced to build tryptophan.
If tryptophan is not free in the environment, the cells must biosynthesize anabolism the tryptophan on its own.
So if no tryptophan is being made, the promoter, operator, structural genes, upstream regulatory gene, and regulatory gene is going through transcription and translation, and produce the regulatory proteins known as the repressor, but the repressor starts off inactivated so it doesn’t bind to operator.

What happens in a repressible tryptophan (trp) operon with tryptophan present

Tryptophan operon is a biosynthetic pathway for building amino acid tryptophan. It is an anabolic pathway, enzymes will be produced to build tryptophan.
When tryptophan is present, it will bind to the inactivated repressor and activate the repressor. The repressor is going to bind to the operator and cause a physical block in the operon, like with no lactose.

What is a mutation

Change in the nucleotide base sequence of an organism’s genome (Could be 1 or 1000s of nucleotides that are changed)

Are lasting changes to mutations rare and why

Yes, they are rare due to the rigorousness of DNA repair machinery but they are almost always deleterious (causing harm/damage)

Do mutations lead to protein having a novel property that improves the ability of an organisms and its descendants to survive and reproduce

No, this is rare (and would take thousands of mutations)

What are the 2 main types of mutations

Point mutations and frameshift mutations

What is point mutations

Most common mutation that affect only 1 or sometimes a few base pairs at a time

What is frameshift mutations

Nucleotide triplets after the mutation displaced such that the reading frame of codons is altered

What is silent mutation

A base gets altered but it does not change the amino acid that is coded.
Effects of mutations are silent and we haven’t changed the amino acid sequence of the protein
(you change a single nucleotide which also changes the mutated mRNA and codon, but it still codes for the same thing)
Ex: changing adenine to guanine. The code used to be UUU coding for Phe but is now changed to UUC, but it still codes for Phe.

What is missense mutation

1 amino acid substituted for another, changing the sequence.
This affects the functionality of the protein
(you do change the amino acid sequence. Kind of like silent mutation but you actually do change the amino acid that the nucleotides codes for)
Ex: in the original DNA, there is a guanine and mutation changed it to a cytosine. It used to be GCA but now it is GGA, coding for glycine

What is nonsense mutation

Changes from regular codon to a stop codon and stops protein growth
The nucleotide is changed that results in a stop codon and it terminates the translation
Ex: an adenine has been changed to a thymine. originally, it coded for UAU and now it codes for UAA a stop codon that prematurely terminates translation at this position.

What is frameshift insertion mutation

A base is inserted into the strand and causes a major change in amino acid sequence and a change in reading frame
Ex: if you add a G to the DNA strand

What is frameshift deletion

A base is deleted causing a major change in amino acid sequence and a change in reading frame
Ex: deleting a G from the DNA strand

What are 2 types of mutagens

Radiation (2 types: high energy and low energy) and chemical mutagens (3 parts: nucleotide analogs, nucleotide-altering chemicals, and frameshift)

What is high energy radiation and an example

Potent (powerful), ionizing radiation induce (causes) breaks in chromosomes.
For example: X-rays

What is low energy radiation and an example

Nonionizing radiation induce (causes) crosslinks of thymine dimers between adjacent thymine bases
Ex: UV light

What are the chemical mutagens

Nucleotide analogs, nucleotide-altering chemicals, and frameshift mutagens

What are nucleotide analogs

Disrupts DNA and RNA replication and causes point mutations

What is nucleotide-altering chemicals in chemical mutagens

Result in base-pair substitution mutations and missense mutations

What is frameshift mutagens in chemical mutagens

Results in nonsense mutations

What are the bacterial DNA repair mechanisms

Direct repair & excision repair (the 2 considered precise mechanism), postreplication repair, SOS response, & error-prone repair

What is direct repair of bacterial DNA repair mechanism (1/2 precise mechanism)

Enzymatic removal of damage, including thymine dimers and alkylated bases

What is excision repair of bacterial DNA repair mechanism (1/2 precise mechanism)

Removal of damaged DNA, followed by synthesis of new strand via opposite (template) strand

What is postreplication repair of bacterial DNA repair mechanism

Retrieval of missing information by recombination, especially when both strands are damaged

What is SOS response repair of bacterial DNA repair mechanism

Induction of about 15 genes after DNA damage or interruption of DNA replication

What is error-prone repair of bacterial DNA repair mechanism

Mechanism of last resort used before a cell dies; fills gaps with random sequences when template information is missing

What is genetic recombination

Exchange of DNA segments that share similar (homologous) sequences

What is recombinants

Cells with DNA molecules that contain new nucleotide sequences

What is vertical gene transfer

Organisms replicate their genomes and pass genes to their descendants

What is horizontal gene transfer

Donor contributes only part of the genome to a recipient, there are 3 types: transformation, transduction, and conjugation

What are the 3 parts of horizontal gene transfer and what is it

Transformation: naked pickup of DNA from environment
Transduction: involves a virus
Conjugation: physical contact by conjugation pili between a donor cell and recipient cell

Who can do genetic recombination and horizontal gene transfer

Humans can do genetic recombination and we are a result of genetic combination from our parents, which is referred to as vertical gene transfer (verticle gene transfer in regards to bacteria is that they just do binary fission, which is asexual reproduction so it means they just make clones)

Microbes can do horizontal gene transfer which is intergenerational, like if I have brown eyes and find someone with blue eyes, I can take my brown eye gene and pop it into their cells. Instantaneously, the blue eyes will change brown so in horizontal gene transfer, by taking the new gene/trait, the recipient can instantaneously transcribe and translate it.

What was Frederick Griffith experiment in 1920

He was studying the possibility of creating a vaccine against pneumococcus, responsible for causing pneumonia and discovered transformation
He used 2 strains of pneumococcus: a living encapsulated virulent/toxic cell strain (S) and a living non-virulent no capsule cell strain (R). He injected mice with the S strain and it died. He injects mice with R strain and it survives. So he wants to know if it is a toxin that the encapsulated bacteria produces that causes the deaths of pneumonia → he grows the culture again and heart its up so that what is left is only the toxin if present, He injects the toxin into the mice and it survives so no toxin is present. → now he takes the heat-treated bacteria and rough strain (both where mice lives) nd combine it into a same test tube and mice dies. He kept repeating this and notice he put in a rough strain but no capsule but got out a smooth strain with capsule. He concludes there must be a transformation factor and something must be given by the heat-treated cells that is picked up by rough cell to then transform into the smooth cell (the one that kills the mice).
During this time, they did not know genetic material was DNA so in reality, when the heat-treated cells died, their DNA was released out of the cell and into the medium, and the rough cells that were alive in the media were able to consume the gene for capsule production/glycocalyx

What is transformation

The uptake of DNA by a cell from the environment; DNA is often left behind by dead cells

What are cells that take up DNA in transformation

Competent cells. Competency results from alterations in the cell wall and the cytoplasmic membranes that allow DNA to enter a cell. If DNA segment codes for a novel trait, the recipient cell can express the new characteristic.

Based on Frederick Griffith work, what is Avery et al find

That the transforming agent was DNA; one of conclusive pieces of evidence that DNA is genetic material

What is transduction

Transfer of DNA from donor cell to a recipient cell through a replicating virus (bacteriophage)

What is generalized transduction

Transducing phage carries random DNA segment from donor to recipient (if DNA segment codes for a novel trait (penicillin resistant), the recipient cell can express the new characteristic)

What is specialized transduction

Only certain donor DNA sequences are transferred (important mechanism by which bacterial toxin genes are transferred between cells & can be used for research purposes to insert genes into cell intentionally)

What does transduction/generalized transduction teach us

If DNA segment codes for a novel trait (penicilin resistant), the recipient cell can express a new characteristic.

What is bacterial conjugation

Transfer of DNA between bacterial cells that are in physical contract with each other

What does DNA transfer require and how is it mediated in bacterial conjugation

By the formation of conjugation pili (sex pili) or special adhesions on the cell surface (some Gram-positive strains)

What gives a bacterial conjugation the ability to synthesize a pillus for conjugation

Conferred by F plasmid (fertility factor)
Involves a donor and recipient cell. Donor cell contain F plasmid/F plus and recipient cell does not contain this so it is called F minus. Physical contract occur between the 2 cells by the pili and the cell containing F plasmid starts replicating the plasmid and sends it through at the end of this process so that now both cells have F plasmid.

How do you differentiate based on the 3 different types of horizontal gene transfer

Based on transformation which is the naked pickup of DNA from the environment, versus transduction that involves a virus as the mediator, versus bacterial conjugation where 2 cells have physical contact