gene expression and protein synthesis

Question 1

3 types of rna for protein synthesis

Answer 1

messenger-used as a template to make proteins (3-5% or total rna)

transfer-brings amiino acids to mrna and ribosomes for assembling proteins (15% of total rna)

ribosomal-makes up structural component of ribosomes (80% of totoal rna)

Question 2

How does transcription initiation differ between eukaryotes and prokaryotes?

Answer 2

Eukaryotes:

Use a TATA box (~25–35 bases upstream of the start site) in the promoter.

Require transcription factors (proteins) to help RNA polymerase II bind to DNA.

RNA polymerase II cannot bind to DNA directly; it needs these helpers.

Prokaryotes:

Use -10 and -35 sequences (each 6 bases long) in the promoter.

RNA polymerase binds directly to these sequences without transcription factors.

Question 3

What happens during the elongation step of transcription?

Answer 3

RNA synthesis occurs in the 5’ to 3’ direction (complementary to the DNA template).

RNA polymerase reads the template strand in the 3’ to 5’ direction.

As RNA polymerase moves, it untwists the DNA helix (~10–20 bases at a time).

RNA polymerase has a proofreading mechanism to correct errors in base pairing.

Question 4

What happens during RNA processing in eukaryotes, and how does it differ from prokaryotes?

Answer 4

Eukaryotes:

-5’ Cap: Adds 7-methyl guanosine triphosphate to protect RNA and help ribosome binding. (helps attach to ribosome and protects from degredation by enzymes like rnases)
-Poly-A Tail: Adds 100–250 adenines at the 3’ end for RNA stability and export. (helps transport out of nucleus and protection)
-Splicing: Removes introns (non-coding sequences) and joins exons (coding sequences) using the spliceosome (snRNAs and proteins).

Prokaryotes:
RNA is ready for translation immediately—no capping, tailing, or splicing needed.

Question 5

What are introns

Answer 5

Introns are non-coding sequences (“intervening sequences”) in the primary RNA transcript (pre-RNA). They do not code for protein and need to be spliced out. Exons, the coding sequences (“expressing sequences”), are already present in the pre-RNA. The splicing process, which occurs in the nucleus, is carried out by the spliceosome (a complex of small nuclear RNAs and over 300 proteins). After the introns are removed, the ends of the exons are re-attached, forming the final RNA that consists only of exons, which code for protein.

Question 6

More than one codon is used for most amino acids: the genetic code is
“degenerate”

Answer 6

This means that it is not possible to take a protein sequence and deduce exactly the
base sequence of the gene it came from

Question 7

possible codons, but code for # different amino acids

Answer 7

64 possible codons (three nucleotide sequence), 20 different amino acids

Question 8

start and stop codons

Answer 8

1 start aug-methionine
3 stop-5’-3’uga, uag, uaa

Question 9

What is the structure and function of transfer RNA (tRNA)?

Answer 9

Transfer RNA (tRNA) are short RNA sequences that fold into a characteristic cloverleaf pattern. Each tRNA has an anticodon, which is a set of 3 bases that pairs with the 3 bases of the codon on mRNA during translation. Additionally, each tRNA has its corresponding amino acid attached to the 3’ end, a step that occurs during the activation phase of translation. This allows tRNA to bring the correct amino acid to the ribosome for protein synthesis.

Question 10

What are the three key sites on the ribosome and their functions during translation?

Answer 10

Peptidyl-tRNA site (P site):
This site holds the tRNA that carries the growing polypeptide chain. It is where the amino acids are added to the chain during protein synthesis.

Aminoacyl-tRNA site (A site):
This site binds the tRNA that carries the next amino acid to be added to the polypeptide chain. The codon on the mRNA pairs with the anticodon on the tRNA here.

Exit site (E site):
This site is where the empty tRNA (after it has delivered its amino acid) exits the ribosome, ready to be recharged with another amino acid.

Question 11

activation in translation

Answer 11

During the activation step of translation, the amino acid is attached to the 3’ end of the corresponding tRNA. Once the amino acid is attached, the tRNA is considered “charged” and ready to deliver the amino acid to the ribosome for protein synthesis.

Question 12

initiation in translation

Answer 12

During the initiation stage of translation, the following steps occur:

The small ribosomal subunit binds to the 5’ end of the mRNA, specifically to the ribosomal binding site (a sequence of nucleotides upstream of the start codon) with the help of initiation factors.
A tRNA carrying methionine binds to the start codon (5’-AUG-3’) on the mRNA.
The large ribosomal subunit is then brought in with the help of GTP, forming the complete initiation complex, ready for protein synthesis to begin.

Question 13

What happens during the termination stage of translation?

Answer 13

Translocation exposes a stop codon in the A site.
A release factor binds to the stop codon in the A site (since there are no tRNA molecules with anticodons for stop codons).
The release factor binding triggers the cleavage of the polypeptide chain from the tRNA in the P site.
The polypeptide is released, and the tRNA is also released.
The two ribosomal subunits and mRNA dissociate from each other, a process catalyzed by GTP.

Question 14

How are transcription and translation different in bacteria and eukaryotes?

Answer 14

In bacteria, transcription and translation are coupled and occur simultaneously in the cytoplasm. As soon as the mRNA is transcribed, ribosomes begin translating it into protein.
In eukaryotes, transcription and translation are spatially and temporally isolated. Transcription occurs in the nucleus, and the mRNA must be processed and transported out of the nucleus before translation occurs in the cytoplasm.

Question 15

monocistronic mrna vs polycistronic

Answer 15

• Monocistronic
  – One mRNA molecule translates for only one type of protein
  – Refers to eukaryotic mRNA. 1 translation start site
• Polycistronic
  – One mRNA molecule translates for more than one type of protein
  – Refers to prokaryotic mRNA. mulitple transaltion start sites

Question 16

point mutaions

Answer 16

change in just one base. two types: nucleotide pair substitutions (3 subcategories) and insertions or deletions.

nucleotide pair substitutions:
-nonsense=code for a stop codon, usually ends up in nonfunctional protein
-missense=codes for a different amino acid
-silent=no chnge bc of redudency in coding

insertions/deletions result in disasterous effect. frameshift mutation

Question 17

What are the main points where gene regulation occurs?

Answer 17

Transcription: The primary regulation point for most genes, determining whether a gene is transcribed or not.
Chromatin Accessibility: The structure of chromatin can be regulated (more open chromatin makes genes more available for transcription).
RNA Processing: Splicing, capping, poly-A tail addition, and RNA exit from the nucleus can be regulated.
RNA Stability: The lifetime of an mRNA in the cytosol affects how many proteins can be made from it.
Translation: Protein levels can be regulated by increasing or inhibiting translation.

Question 18

What controls the lac operon, and what are the roles of its genes?

Answer 18

The lac operon is controlled by two regulatory proteins:

Lac repressor: Detects lactose and can bind to DNA to inhibit transcription.
Catabolite activator protein (CAP): Detects glucose and promotes transcription when glucose is low.
The operon produces polycistronic RNA (a single mRNA that codes for multiple proteins), consisting of three genes:

lacZ: Codes for beta-galactosidase, which splits lactose into glucose and galactose.
lacY: Codes for lactose permease, a transport protein that pumps lactose into the cell.
lacA: Codes for thiogalactoside transacetylase, which acetylates lactose.

Question 19

What is the role of the lacI gene in the lac operon?

Answer 19

The lacI gene, located near the lac operon, codes for the lac repressor protein, which is essential for controlling the lac operon.

Key points about lacI:

It is expressed constitutively (always on, but at a low level).
It produces a separate mRNA, different from the lac operon’s polycistronic RNA.
The lac repressor protein binds to the operon’s operator region to regulate transcription.

Question 20

What is the difference between cis-regulatory elements and trans-regulatory proteins?

Answer 20

Cis-regulatory elements are non-coding DNA sequences that help regulate nearby genes. They are not transcribed into mRNA or made into proteins. Example: operator region in the lac operon. (includes promoters and operators)

Trans-regulatory proteins are coding proteins made from genes. They are transcribed into mRNA and translated into proteins, which then regulate gene expression by binding to cis-regulatory elements. Example: lac repressor protein. (includes repressor)

Question 21

What is the role of the lac repressor and where does it bind?

Answer 21

The lac repressor binds to the operator region of the lac operon, which prevents RNA polymerase from binding to the promoter and starting transcription of the lac genes when lactose is absent.
The promoter is where RNA polymerase binds to begin transcription, but the repressor blocks this process by binding to the operator, stopping gene expression.

-la repressor can bind to either operator or lactose. if theres no lactose present itll bind to operator, blocking polymerase from transcribing leading to no genes encoding proteins like beta-galactosidase

Question 22

What is an inducer in gene regulation?

Answer 22

inducer is a molecule that binds to the repressor protein and changes its shape, preventing the repressor from binding to the operator region.
This allows RNA polymerase to bind to the promoter and transcribe the genes, activating gene expression.
In the case of the lac operon, lactose acts as the inducer, which binds to the lac repressor, allowing the lac genes to be transcribed when lactose is present.

Question 23

mutations in regulatroy proteins/elements:

Answer 23

Null Mutation (lacI gene): DNA sequences with this mutation have completely lost their
normal activity
o In protein-coding genes, this means no protein is produced
o In regulatory genes, this means that regular binding sites are non-functiona

Constitutive Activity (Oc mutation): specific to the operator region
o Constitutively active operator regions always block the binding of repressor protein
to the operator region
o This results in transcription of the operon whether or not lactose is present,
because the repressor is unable to block RNA polymerase from binding to the
promote

Superrepressor (Is mutation): Super-repressor genes produce special repressor proteins, which can still bind to
the operator but not to lactose

so either gene messed up (repressor or actual proteins arent produced), repressor proteins come out fucked and cant bind to inducer at all only to operator, cant bind to operator and operator itself is fucked

Question 24

What happens when glucose is present and lactose is absent in the lac operon?

Answer 24

No transcription of the lac operon occurs.
The lac repressor binds to the operator, preventing RNA polymerase from transcribing the operon.
cAMP levels are low because glucose levels are high. This makes CAP inactive, so it cannot bind to the CAP site on the DNA.
The lack of active CAP reduces the binding affinity of RNA polymerase to the promoter, further preventing transcription.

Question 25

What is a merodiploid?

Answer 25

A merodiploid is a bacterium that has two copies of part of its chromosomal DNA: one on the chromosome and one on a plasmid, making it partially diploid for that specific section.

Question 26

What is the difference between positive and negative regulation in operons, using the lac and trp operons as examples?

Answer 26

Lac Operon: Positive regulation. It is activated by an effector (lactose) and requires the CAP protein to help turn on gene expression when glucose is low.
Trp Operon: Negative regulation. It is inhibited by an effector (tryptophan) that binds to a repressor and blocks gene expression when tryptophan is abundant.

Question 27

What is attenuation in the trp operon?

Answer 27

Attenuation is a process that controls transcription of the trp operon based on tryptophan levels.
When tryptophan is high, the ribosome moves quickly and causes a hairpin structure in the mRNA that stops transcription early.
When tryptophan is low, the ribosome moves slowly, allowing a different hairpin structure to form that lets transcription continue, making more tryptophan.