Exam 3: Microbial Genetics I- Transcription and Translation

Question 1

Describe the basic flow of information in the expression of a gene, naming each of these key processes.

Answer 1

Transcription forms and RNA copy of a gene then translation produces a polypeptide based on the information in this RNA copy of the gene.

Question 2

List the three key steps in transcription.

Answer 2

1) Initiation of transcription
2) Elongation of the RNA transcript
3) Termination traqnscription

Question 3

At what DNA site does transcription begin?

Answer 3

Promoter

Question 4

3 parts of a gene

Answer 4

Promoter: beginning of gene, site where transcription begins.

Terminator unit: portion of gene copied into RNA, contains information dictation production of a specific protein, begins within the promoter and ends near the terminator.

Terminator: end of a gene, site dictating end of transcription.

Question 5

Can a particular gene be transcribed by only a single enzyme at a time? Explain.

Answer 5

No, a single gene can be simultaneously transcribed by multiple RNA polymerase enzymes. Once another RNA polymerase has cleared the promoter, another RNA polymerase can bind.

Question 6

Describe how transcription begins in bacteria.

Answer 6

RNA polymerase attaches nonspecifically to DNA. Travels until it reaches promoter sequences. Enzyme that unzips the DNA molecule at the promoter.

Promoter recognition is dependent upon a polypeptide subunit of RNA polymerase termed sigma factor. Different RNA polymerases use different sigma factors. There is also variation in promoter sequences. This variation provides some control over transcription.

Question 7

Describe the events of the elongation phase of transcription.

Answer 7

NTPs align with DNA compliments. RNA polymerase links these nucleotides together. Assembly begins ~10 nucleotides downstream of promoter, energy provided by high energy phosphate bonds. RNA synthesis continues through genes, only one of the DNA strands is transcribed.

Question 8

By what means is energy supplied to drive the polymerization of RNA during transcription? How does this compare to the polymerization of DNA?

Answer 8

The energy source is similar, they both come from high energy phosphate bonds.

Question 9

How fast are nucleotides added to a growing strand of RNA during transcription? How does this compare to the speed of DNA synthesis?

Answer 9

Slower compared to speed of DNA synthesis at 50 nucleotides per second instead of 500-1000.

Question 10

Differences between DNA synthesis and RNA synthesis:

Answer 10

• RNA polymerase unwinds the DNA-no helicase is needed
• No primer is required
• Only one DNA strand is copied
• Polymerization is much slower
  ~50 nucleotides per second, not 5000 – 1,000
• Ribonucleotides incorporated, not deoxyribonucleotides
• Uracil is used in place of thymine
• Less efficient proofreading

Question 11

Briefly describe rho-dependent termination.

Answer 11

Dependent upon the termination of protein “Rho”. Binds to an RNA sequence near the 5’ end of the transcript, moves towards the RNA polymerase moving faster than the polymerase. Rho pushes between RNA polymerase and the DNA pushing them apart.

Question 12

Briefly describe rho-independent termination.

Answer 12

Also called “self-termination”. RNA polymerase transcribes a terminator sequence. GC- rich segment followed by A-rich segment. RNA polymerase slows while transcribing GC-rich segments as the three H-bonds are hard to break.

Complementary regions in the transcribed RNA bind to each other resulting in a hairpin loop formed by RNA. Weak binding between RNA and DNA when transcribing the A-rich portion cannot withstand the tension so the rNA polymerase is released and transcription ends.

Question 13

In what key ways does transcription differ between bacteria and eukaryotes?

Answer 13

Eukaryotic transcription takes place in the nucleus. Eukaryotic nucleoid.

Eukaryotes have 4 kinds of RNA polymerase.

Eukaryotes have transcription and elongation factors.

mRNA is processed in eukaryotes multiple ways prior to translation.

Question 14

Eukaryotes have four kinds of RNA polymerase

Answer 14

1) One type transcribes mRNA

2)One type transcribes the major rRNA gene

3)One type transcribes tRNA and smaller rRNAs

4)Mitochondria use a fourth type of RNA polymerase

Question 15

What types of organisms process RNA following transcription but preceding translation?

Answer 15

Eukaryotic organisms

Question 16

Name each of the three processes involved in RNA processing.

Answer 16

Capping
Polyadenylation
Splicing

Question 17

Capping

Answer 17

Modified “G” is added to the 5’ end of the transcript. Added “backwards” compared to other nucleotides and occurs when RNA is ~30 nucleotides long.

Question 18

Polyadenylation

Answer 18

100-250 “A” nucleotides added to the 3’ end of the transcript. No DNA template is used.

Question 19

Splicing

Answer 19

Pre- mRNA has non-coding “introns” and coding” exons”. Introns are intervening sequences and exons are exported and expressed sequences. Spliceosomes remove introns and covalently join the exons. Intron removal accomplished by spliceosomes which act as ribosomes. Function mRNA produced and excised introns degraded.

Single gene can encode multiple polypeptides accomplished through alternative splicing. Results in the formation of different polypeptides.

Question 20

Are all three of the processes catalyzed by enzymes? Explain.

Answer 20

No just the splicing.

Question 21

What are introns and what are exons?

Answer 21

Introns are intervening sequences. Exons are exported and expressed sequences.

Question 22

How does pre-mRNA differ from mRNA?

Answer 22

Modified “G” added to the 5’ end, “A” nucleotides added to the 3’ end, introns removed and exons spliced together.

Question 23

Are all RNAs translated? Explain.

Answer 23

No, only mRNA translated into protein the others are functional as RNA molecules

Question 24

Translation

Answer 24

Uses information in the sequence of mRNA bases

Produces a polypeptide with a specific sequence of amino acids

Question 25

Amino acid relevance to translation

Answer 25

Polymerized to form a polypeptide. Peptides consist of linear chains of amino acids, 20 different amino acids arranged in different orders to different lengths to make different polypeptides.

Question 26

mRNA relevance to translation

Answer 26

Temporary copy of a gene. Divided into units called codons. Consist of 3 consecutive nucleotides, each codon corresponds to a specific amino acid. Relationship between codons and amino acids is termed “genetic code”.

Question 27

Genetic code relevance to translation

Answer 27

“Language” of the mRNA. Relationship between codons and amino acids. 64 different codons, all 20 amino acids are represented. Universal code, same in all living things. Slight differences in some species. Also have start and stop codons.

Question 28

tRNA relevance to translation

Answer 28

Interact with mRNA and provide amino acids. Short segments of RNA involved in the transfer of appropriate amino acids to grow a polypeptide chain. Numerous different tRNA correspond to different codons. Folds back on itself to form three main hairpin loops. Contains anticodon which is a nucleotide triplet complementary to mRNA codon. Contains amino acid attachment site “acceptor stem”.

Question 29

Ribosome relevance to translation

Answer 29

Cellular organelle responsible for covalently linked amino acids. Covent Cellular organelles serving as the site of translation, present in many copies within a cell. large subunit and small subunit. Composed of rRNA and polypeptides. facilitate the specific interactions between codons and anticodons. Catalyze the formation of covalent bonds between amino acids in he growing polypeptide chain.

Question 30

What term is used to describe three consecutive nucleotides in an mRNA molecule? Describe the importance of these units.

Answer 30

Codon, each codon corresponds to a specific amino acid.

Question 31

Describe the specific interactions between an mRNA and a tRNA.

Answer 31

tRNA molecules are responsible for matching amino acids with the right codons in mRNA. Each tRNA molecule has two different ends, one that binds to a unique amino acid and one that binds to the appropriate mRNA codon. In translation, tRNAs transport amino acids to the ribosome that bind with their complementary codons.

Question 32

How does a stop codon differ from the other 61 codons?

Answer 32

stop codons signal the termination of this process by binding release factors, which cause the ribosomal subunits to disassociate, releasing the amino acid chain

Question 33

What is the first codon read during translation?

Answer 33

AUG- Met or start

Question 34

Three stages of translation

Answer 34

Initiation
Elongation
Termination

Question 35

What is the first amino acid incorporated into every polypeptide chain? How does this differ between bacteria and eukaryotes?

Answer 35

In eukaryotes its methionine and in bacteria its formylmethionine.

Question 36

The genetic code is degenerate. What does this term mean?

Answer 36

Multiple codons code for the same amino acid.

Question 37

Do all organisms have essentially the same genetic code? Why is this the case? Why is this important in terms of horizontal gene transfer?

Answer 37

Essentially the same genetic code for all organisms, due to shared genetic ancestry. Make horizontal gene transfer possible since everyone uses the same code.

Question 38

Are there truly only twenty different amino acids? Explain.

Answer 38

Because amino acids can be arranged in many different combinations, it’s possible for your body to make thousands of different kinds of proteins from just the same 21 amino acids.

Question 39

Describe the nature and structure of a tRNA. How do the different tRNAs differ from each other?

Answer 39

tRNA are short segments of RNA involved in the transfer of amino acids to a growing polypeptide. They differ in their anticodon sequence.

Question 40

Describe the structure and composition of a ribosome. Which component constitutes the bulk of the mass of a ribosome?

Answer 40

Composed of two subunits, large and small. Each subunit composed of rRNA and polypeptides. 1/3 mass is protein ⅔ of mass is rRNA.

Question 41

Describe process of translation

Answer 41

1) initiation: a small ribosomal subunit attaches to mRNA at a ribosome-binding site. Located just upstream of the start codon, scans down the mRNA until encountering the start codon.
Small subunit also binds to an initiator tRNA which can occur either before or after binding to the mRNA.
Initiator tRNA H-bonds to the start codon and the ribosome’s “P site”, large ribosome subunit attaches.

2) Elongation: Begins with initiator rRNA in ribosomes “P site”. tRNA binds to codon in ribosomes “A site”
Ribosomes has 3 sites A, P, and E. P site is initially where initiator tRNA binds, P side is back at the AUG, and A site is just downstream of P site.
Ribosome moves on codon down the mRNA chain.
Start codon exposed as translation progress, another ribosome attach behind the firs so multiple ribosomes can simultaneously translate an mRNA.

3) Termination: no tRNA recognize stop codons. Stop codons are recognized by “release factors”
Larger ribosomal unit is modified, another ribosome ribozyme i s activated. Peptide is severed from the tRNA.

Question 42

Describe how translation initiates in bacteria, and contrast this with initiation of translation in eukaryotes. How are they the same, and how are they different?

Answer 42

• Initiation of translation involves binding of the small ribosomal subunit to the 5’ guanine cap
• The first amino acid in eukaryotic polypeptides is methionine, not formylmethionine
• Some eukaryotic ribosomes are attached to the RER
• Translation in archaea is similar to that in eukaryotes

Question 43

Describe the importance of the P site, the A site, and the E site of a ribosome.

Answer 43

Stages of translation (article) | Khan Academy
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).

Question 44

Can a given mRNA be translated by only a single ribosome at a time? Explain.

Answer 44

Messenger RNAs can be translated simultaneously by several ribosomes in both prokaryotic and eukaryotic cells. Once one ribosome has moved away from the initiation site, another can bind to the mRNA and begin synthesis of a new polypeptide chain.

Question 45

Must transcription be complete before translation begins? Is the answer the same in bacteria and in eukaryotes? Explain.

Answer 45

In prokaryotes, translation of a transcript begins before the transcript is complete, due to the proximity of ribosomes to the new mRNA molecules. In eukaryotes, however, transcripts are modified in the nucleus before they are exported to the cytoplasm for translation.

Question 46

What is a polycistronic mRNA? Are they present in both bacteria and eukaryotes? Explain.

Answer 46

Multiple protein-coding units are contained within a single mRNA. Each gene is translated separately. Not possible in eukaryotes where only a single cap exist, only possible in bacteria.

Question 47

Why is the use of antimicrobials targeting bacterial ribosomes useful?

Answer 47

Because they are distinct from eukaryotic ribosomes, antimicrobials that started these differences leave eukaryotic cells alone.

Question 48

By what mechanism does streptomycin inhibit translation?

Answer 48

Interfere with codon reading by changing the shape of the small subunit. Because they read the codons wrong they bring in the wrong tRNA and incorporate the wrong amino acids into the polypeptide chain. Protein is still made but not functioning due to incorrect amino acids.

Question 49

By what mechanism does chloramphenicol inhibit translation?

Answer 49

Blocks the enzymatic site of the ribosome’s large subunit. Binds to the ribosomes in a place that is important for catalyzing the formation of covalent bonds. Bonds between amino acids are not formed.

Question 50

By what mechanism does doxycycline inhibit translation?

Answer 50

Bind to the large subunit in the area where transfer rRNA typically comes in, the “A site”. tRNA is blocked from coming in a binding so synthesis of the polypeptide is halted.

Question 51

By what mechanism do clindamycin and erythromycin inhibit translation?

Answer 51

Bind to a large subunit and prevent it from sliding down the messenger RNA. It stops more tRNA form being added and incorporation of amino acids stops.

Question 52

By what mechanism do antisense nucleic acids inhibit translation?

Answer 52

Block attachment to mRNA.

Question 53

By what mechanism does mupirocin inhibit translation?

Answer 53

Binds to isoleucyl-tRNA synthetases in Gram-positive bacteria. Does not bind to eukaryotic enzymes, isoleucine cannot be “loaded” onto tRNAs, translation cannot proceed past isoleucine codons.

Question 54

Where does regulation occur?

Answer 54

Regulation occurring at the transcription level is most common however it can also occur at the translation level.

Question 55

Operon

Answer 55

Key unit of gene regulation in bacteria. Consists of a group of genes with related functions. e.g. genes involved in the synthesis of a particular amino acid. e.g genes involved in the metabolism of a particular sugar.

Regulated as a group in response to specific environmental conditions. Transcribed together in a polycistronic mRNA.

Question 56

Briefly describe the structure of a generic operon, describing the function of each component.

Answer 56

Promoter: site which RNA polymerase binds to begin transcription

Structural genes: enzymes, transport proteins, etc.

Operator: regulatory element downstream of promoter. Repressor protein can bind to operator & block transcription.

Regulatory gene: (controlled by its own promoter). Produces protein that can blind to the operator. Always on.

Question 57

Is there a 1:1 correspondence between genes and mRNAs in an operon? Explain.

Question 58

MicroRNAs “miRNAs”

Answer 58

Untranslated single-stranded RNA molecules, ~22 nucleotides in length, and transcribed by eukaryotic cells.

Join with regulatory proteins to form a miRNA-induced slicing complex (miRISC), bind to mRNA with complementary sequence, two possible outcomes miRISC cuts the mRNA and mRNA is rendered useless or remains bound and blocks mRNA from ribosomes mRNA is rendered useless.

Question 59

siRNAs

Answer 59

Short, untranslated, double-stranded RNA molecules. Similar to miRNA in size.

Hairpin loop is cut by the enzyme dicer to generate a double-stranded siRNA. siRNAs join RISC proteins to form siRISC, binds to complementary mRNA, tRNA, or DNA. SiRISC binds to and cuts target nucleic acid resulting in gene splicing.

Question 60

Riboswitches

Answer 60

RNA molecule that helps regulate translation in bacteria and eukaryotes. Alters shape in response to environmental conditions. One shape allows translation, one does not. Some mRNAs can themselves act as riboswitches, under specific conditions folds to either favor or block translation.

Question 61

Is there a 1:1 correspondence between genes and mRNAs in an operon? Explain.

Answer 61

No, operons work on a cluster of genes since related genes are normally clustered on the chromosome.

Question 62

Describe the basic relationship between the multiple gene products encoded by an operon.

Answer 62

They normally work together as part of the same metabolic pathway.

Question 63

How does a repressible operon differ from an inducible operon? Can both exist in either an “on” or an “off” state?

Answer 63

Inducible operon: always “off” but can be turned “on”. Usually not transcribed. Can be activated by molecules termed “inducers”. Example; Lac Operon.

Repressible operon: Always “on” but can be turned “off” . Transcribed continually. Can be deactivated by molecules termed “repressors”. Example; Trp Operon

Question 64

How is a repressible operon like an inducible operon?

Answer 64

Both have the ability to be in the on or off position.

Question 65

What types of genes are commonly governed by a repressible operon?

Answer 65

Anabolic pathways.

Question 66

What types of genes are commonly governed by an inducible operon?

Answer 66

Catabolic pathways whose polypeptides are not needed unless a specific nutrient is available. Production of virulence proteins by pathogens.

Question 67

What is a repressor? In what states can a repressor exist? What governs the current state of a repressor?

Answer 67

Molecules that can deactivate a repressible operon. Can be inactive or active. Normally inactive so operon is being expressed normally.

Question 68

What is an inducer?

Answer 68

Signaling molecule that activates an inducible operon.

Question 69

Briefly describe the state of the lac operon in the absence of lactose.

Answer 69

Inducible operon, involved in the transport and catabolism of lactose. Normally not transcribed.

Default state is off, repressor bound to operator blocking the promoter. If glucose is present CAP is not bound to the CAP-binding site.

Question 70

In what way is the lac operon affected by the presence of lactose?

Answer 70

Induced by lactose (the inducer). Lactose is not always present so it stays off, when present turns on.

Lactose converted to allolactose which binds to repressor protein changing its shape. Repressor can no longer bind to the operator, opening it up to RNA polymerase to bind to promoter and transcribe genes.

Once lactose has been depleted, the repressor is no longer inactivated as there is no longer allolactose. Functional repressor protein turn operon “off”

Question 71

In what way is the lac operon affected by the presence of glucose?

Answer 71

It is kept “off”. Glucose is more easily metabolized than lactose. The cells preferentially metabolize the most efficient sugar (glucose). CAMP is not accumulating so CAP is not bound, CAP-binding site is open.

Question 72

Briefly describe the state of the lac operon in the presence of both glucose and lactose.

Answer 72

“off” it is going to use glucose instead of lactose. CAP is not bound but repressor protein is unbound.

Question 73

Briefly describe the state of the lac operon in the presence of lactose but the absence of glucose.

Answer 73

“on” will use lactose until it is depleted or there is glucose present.

cAMP accumulates when glucose is absent. cAMP binds to CAP and changes its shape. CAP then binds to a CAP binding site. Only when CAP is bound can RNA polymerase bind efficiently. Allolactose binds to the repressor unlocking it from the operator.

Question 74

OC mutant:
IS mutant:
I- mutant:

Answer 74

OC mutant: The mutant operator cannot be bound by the repressor.

IS mutant: The “super-repressor” cannot bind to the inducer.

I- mutant: The repressor gene encodes a protein with no functional activity.

Question 75

How would each of the following mutant versions of the lac operon function when both glucose and lactose are absent? When glucose is absent, but lactose is present?

OC mutant: The mutant operator cannot be bound by the repressor.

IS mutant: The “super-repressor” cannot bind to the inducer.

I- mutant: The repressor gene encodes a protein with no functional activity.

Question 76

Describe the state of the trp repressor in the absence of the amino acid tryptophan.

Answer 76

Repressor is off so the operon is on.

Trip operon: repressible operon that contains genes necessary for the biosynthesis of the amino acid tryptophan. Transcribed under normal conditions “on”. Can be repressed under specific conditions when tryptophan is abundant .

Question 77

Describe the state of the trp repressor in the presence of the amino acid tryptophan.

Answer 77

Repressor binds to tryptophan that’s present changing the repressor shape. The repressor then binds to the operator blocking operon function its now “off”.

Tryptophan not present causes the repressor to lift and go back to its inactive shape.

Question 78

Once the trp operon is induced, will it remain “on” forever? Explain.

Answer 78

No, once tryptophan in the environment is depleted. Repressor is deactivated so that production resumes. and operon is active again.

Question 79

Compare the binding of an inducer to the lac repressor and to the trp repressor. How are these interactions alike? How are they different?

Answer 79

Inducer to the lac repressor turns it off and opens the operon up for activity. Induction of the trp repressor turns on so that activity stops and the organisms can use the available tryptophan in the environment.