Genetic code, tRNA structure and regulation of gene expression (L. 8 and 10)

What direction are codons read ? And proteins synthesised?

5’ to 3’

N’ to C’ (carboxyl)

Before the dvpt of what technique was the genetic code determined? How then was it determined?

techniques for isolation of genes and sequencing of DNA

observing the result of artificial RNAs eg the amino acids in the final protein produced

Give a specific example of a codon that was experimentally found to code for a specific amino acid

Poly(U) RNA = phenylalanine into protein. The codon UUU was deduced to encode phenylalanine

What recognises codons? How?

cognate tRNAs (cognate = correct pairing)

through codon to anticodon base pairing

What is the role of charging tRNAs in codon recognition?

allows connection to the appropriate amino acid

What bonds are formed during mRNA reading?

Peptide bonds between aminoacyl and peptidyl groups attached to adjacent tRNAs on the ribosome

Why is the genetic code called degenerate?

multiple different codons code for one same amino acid

How many codons are there? How many encode a specific amino acid?

64

61, 3 code for termination

Which amino acids are encoded by unique codons?

Met and Trp

What effect can the change of the third nucleotide of a codon cause?

Synonymous codons

What are two ways in which synonymous codons are recognised?

• by different isoacceptor tRNAs that are charged with the same amino acid

• codons that differ at the 3^rd base can be recognized by the same tRNA by wobble base-pairing.

Which nucleotides can Isoine pair with?

U, A, C

Which nucleotides can Guanine in tRNA base pair with through anticodon association?

C or U in 3rd position

At which position do many tRNAs have the nucleotide inosine (I)?

at the 1^st position of the anticodon.

How do tRNAs function in base pairing?

Through the association of anticodons to the codons read

Which nucleotides are purines?

I, A, G

What does post transcription modifications allow tRNAs to do?

To recognise more than one type of codon

What is the secondary structure of tRNAs called? What is the relative position of the 5’ and 3’ ends?

• cloverleaf structure

• they are drawn together

Where does an amino acid attach to a tRNA?

to the 3’ hydroxyl group of the 3’ terminal A nucleotide

What % of nucleotides in tRNA are post transcriptionally modified?

10

What is the tertiary structure of tRNAs? What does it allow?

flat, right sided L-shape

binding site

What is coaxial stacking? How does this structure affect tRNAs?

• short helices and stacked on top of each other like coins

• resulting in an extended helical structure that increases its thermodynamic stability

What is the relative position of the anticodon loop and the aminoacyl group?

positioned at opposite ends of the molecule = flat, L-shaped molecule

Where does base pairing in tRNAs occur? What is the function of each arm?

between the D and ΤΨC loops

acceptor stem stacked on the TΨC arm; anticodon is stacked on the D arm

What is tRNA charging? What is it mediated by? Does it require energy?

process of attaching an amino acid to a transfer RNA

aminoacyl-tRNA synthetases

yes, ATP

What is required to charge isoacceptor tRNAs?

A single enzyme charges all isoacceptor tRNAs

How is the amino acid linked to tRNA? By which part of the amino acid?

• by an ester linkage

• between the carboxylic acid group of the amino acid and the 3’ hydroxyl group of the terminal nucleotide.

What environmental factors can modify gene expression? (5)

temperature, nutrients available, toxins, stress, light

What are genes that are constitutively expressed? When are they expressed?

house-keeping genes. Always bc they are independent from env.

What does the regulation of gene expression allow?

• energy conservation

• avoiding waste

• cell differentiation and specialisation

• one same genome = 200 different cells and a huge variety of RNAs and therefore proteins

How does autoregulation of genes occur?

negative feedback

Compare cis and trans gene regulation. How do they interact?

cis is a regulatory sequence within the gene, trans in a protein or RNA that carries a regulatory sequence to act upon another gene

they work in cooperation, trans recognises and activates cis sequences

What are the 4 levels at which genes can be regulated?

• transcription

• translation

• post translation

• RNA processing (in eukaryotes) - alternative splicing

Can different splicing patterns emerge from one same RNA depending on location, spliceosome etc?

What type of regulation is this?

yes, this is alternative splicing, can occur with the recognition of different splice sites

gene regulation at an RNA processing level (only in eukaryotes)

At what level are most genes regulated and why?

Transcriptional level bc avoids waste, if no protein needed, no RNA needed so none is made

What kind of control is a gene under when transcription factors upregulate gene expression? What would this kind of factor be called?

positive control

activator

When under negative control, do RNA Polymerases still bind to the gene sequence?

yes, they just don’t transcribe/are inactive

What kind of control is a gene under when transcription factors down regulate gene expression? What would this kind of factor be called?

negative control

repressors

What kind of molecule is responsible for increasing a transcription factor’s activity? What are the 2 pathways by which this can be done?

a trans acting inducer

either stimulating activator or inhibiting a repressor

What kind of molecule is responsible for increasing a transcription factor’s activity? What are the 2 pathways by which this can be done?

a trans acting corepressor

either stimulating repressor or inhibiting activator

What would an induced gene under negative control mean?

inducer molecule will inhibit repressor = negative control = expression gene ie inducing

What would a repressed gene under negative control mean?

repressor will inhibit activator = negative control = no expression of gene ie repression

What would an induced gene under positive control mean?

inducer molecule will stimulate activator = positive control = expression of gene ie inducing

What would a repressed gene under positive control mean?

repressor molecule will stimulate repressor = positive control = expression of gene ie repression

What is lac operon?

set of genes in bacteria that control the uptake of lactose

What are the three genes encoded in lac operon?

lacZ, lacY, lacA

Which enzymes do the lac operon genes respectively code for?

lacZ: beta galactosidase

lacY: lactose permease

lacA: galactoside acetyltransferase

Which transcription factor independently controls the expression of the lac operon genes? Is it a repressor or inducer gene?

lacI

repressor

What are the 2 steps by which lacI represses lac operon’s expression?

• bound to lacO operator sequences

• blocks RNAP activity ie RNA transcription

How is lacI inhibited by lactose? Does that make lactose an inducer or repressor?

lactose inhibits lacI repression = gene expression

an inducer using negative control

Why is the lac operon gene only activated in presence of lactose and absence of glucose?

glucose is a preferred source of energy, so

Which transcription factor, along with the inducer cAMP, controls the expression of the lac operon genes? Is it a repressor or inducer gene?

catabolite activator protein (CAP)

inducer

What inhibits cAMP production? How does this effect the activation of lac operon?

glucose

no cAMP = non functional CAP = no stimulation of transcription = gene not induced

In summary, is lac operon inducible? Under which type of control is the gen?

inducible under positive and negative control

At what stage is translation usually regulated? What are 2 situations in which gene expression is down regulated through modified translation?

initiation

integrated stress response (lack of nutrients, infection by virus) and condition specific like ion level can lead to downregulation of genes

How is expression of the mRNA encoding the iron-binding protein ferritin influenced by cellular Fe²⁺ levels? Why is not just always expressed?

responsive to changes in cellular iron levels = gene expression regulation at a translational stage, iron acts as an inducer

iron level would be too high = cytotoxic

What is the most common form of post translational modification = gene expression regulation?

Phosphorylation of serine, threonine or tyrosine residues

Describe phosphorylation of a protein

phosphate group added to alcohol acceptor on each side chain of protein

Is phosphorylation reversible and does is require energy? Which proteins are responsible for phosphorylation of a protein?

yes and yes, protein kinases and protein phosphatases reverse phosphorylation.

How can phosphorylation of a protein be beneficial to a cell’s survival?

allows very quick response to cellular conditions, proteins don’t need to be translated (with previous steps of gene expression + transcripted), but simply modified = modified function

Give an example of how phosphorylation can inhibit translation during initiation

• normally, eIF2 hydrolyses GTP to GDP

• is eIF2 is phosphorylated, its alpha subunit becomes inactive

• this eIF2 p binds to eIF2B very tightly = trap in complex that can’t be recycled = no more GDP = no more initiation ie no more translation