Lecture 9

Question 1

Describe Gene expression

Answer 1

The process of going from DNA to a functional product (typically a protein but can be RNA)

The central dogma is the process of DNA -> protein

Question 2

Describe genotype

Answer 2

An organism’s hereditary information - what proteins we get

Question 3

Describe Phenotype

Answer 3

The actual observable or physiological (expressed) traits, determined by our genotype and its interaction with the environment

Question 4

Describe DNA (deoxyribonucleic

Answer 4

The heritable material that is used to store and transmit information from generation to generation

Double stranded. DNA is very stable - as double stranded, difference in sugars and thymine. Needs to be stable as DNA for a cells is not replaceable

DNA is long lived - last lifetime of cell

DNA have triplets

Question 5

Describe RNA (ribonucleic acid)

Answer 5

A molecule that acts as a messenger to allow the information stored in the DNA to be used to make proteins.

RNA has Uracil (U) instead of Thymine, and is single stranded. Much less stable than DNA - due to single strandedness, difference in ribose sugar and uracil impacts this

multiple types of RNA but we focus on transcription of mRNA nor rRNA or tRNA (they use different polymerases/enzymes - similar processes though)

RNA doesn’t last very long (short-lived)

RNA have codons

(RNA can be functional/do cellular functions like proteins)

Question 6

Describe proteins

Answer 6

Molecules that carry out cellular functions.
Little intricacies of the tertiary structure of the protein dictates the function

Question 7

What are the three main steps of Gene expression

Answer 7

Transcription (of RNA from DNA)
Processing (of the pre-mRNA transcript)
Translation (of the mRNA transcript to a protein

Question 8

What are the three steps of Transcription and where does it occur

Answer 8

Initiation, elongation, termination

Occurs in the nucleus

Question 9

Describe initiation (transcription)

Answer 9

It refers to the assembly of multiple proteins required before transcription can commence:
Genes basically always have a promoter region (can be up to 1,000 of base pairs long, called a eukaryotic promoter) with what is referred to as a TATA box ~ 25 nt upstream (before the gene). The template strand is the one with the A of TATA closest to tip, and is read 3’ to 5’

Several transcription factors including the TATA box binding protein - on of the first - assemble on the TATA box.

This produces a kink in the DNA, causing things to touch each other and enables RNA polymerase II (tear drop shaped) to bind along with more transcription factors, which forms the transcription initiation complex - and so transcription begins.

Question 10

Describe the template strand

Answer 10

It is the strand of DNA that is read by the polymerase and the pre-mRNA is made from

Importantly, it is read 3’ to 5’

Question 11

Describe the coding/nontemplate strand

Answer 11

It is the strand of DNA that is not read/used to form pre-mRNA but it codes for the same bases (other than Uracil) of it

The RNA polymerase goes from its 5’ to 3’ sides

Question 12

Describe elongation

Answer 12

DNA is gradually unwound (10-20 nucleotides exposed at a time by helicase activity) and complementary RNA nucleotides are added to the 3’ end of growing transcript - RNA (they are added to the template strand of DNA from 3’ to 5’ based on the complementary base pairing rules to gradually form a RNA from 5’ to 3’).

As the bases are added they form weak hydrogen bonds between the DNA bases and phosphodiester bonds between each RNA nucleotide (between the 3’ hydroxy and 5’ phosphate groups of nucleotides). Hydrogen bonds form first then phosphodiester.

Double helix reforms as transcript leaves the template strand

Question 13

Describe termination

Answer 13

After transcription of the polyadenylation signal (AAUAAA) - which is downstream of the gene in the terminator region of the DNA - nuclear enzymes release the pre-mRNA, and RNA polymerase then dissociates/disassemble from the DNA. The pre-mRNA transcript is now ready for further processing.

Proofreading/fidelity is less than for DNA replications because mRNA can be made again more easily

Question 14

What is a key difference between DNA replication and transcription

Answer 14

The fidelity (proofreading) is less than for DNA replication

Question 15

What strand is read in transcription and what strand isn’t

Answer 15

Template/non-coding strand (3’ to 5’) is read
Non-template/coding strand (5’ to 3’) isn’t

Question 16

Describe promoter (region)

Answer 16

(located upstream) is responsible for starting transcription

Question 17

Describe the Terminator (region)

Answer 17

(located downstream the gene) stops transcription

Question 18

Describe what 5’ and 3’ mean

Answer 18

5’ means that it is the side with the 5th carbon on ribose sugar, 3’ is side with 3rd carbon

Question 19

What are the three steps of mRNA processing and where does it occur

Answer 19

Capping
Tailing
Splicing.

Occurs in the nucleus/spliceosome

Question 20

Describe capping

Answer 20

A modified guanine nucleotide is added to the 5’ end.

Question 21

Describe Tailing

Answer 21

50-250 adenine nucleotides (polyA) are added to the 3’ end.

Question 22

Describe splicing

Answer 22

Occurs in the spliceosome (a large complex of proteins and small RNAs within the nucleus). Introns are removed from the transcript and exons are re-joined to form mature mRNA (bases and adjacent sequences on introns are recognised and spliced).

Question 23

Describe exons

Answer 23

The coding regions - including UTRs

Exons typically a lot smaller

Question 24

Describe introns

Answer 24

The non-coding regions intervening exons

introns typically much larger

Question 25

Describe UTRs

Answer 25

The untranslated regions at 5’ and 3’ ends (UTRs don’t go on to form protein but still important for forming protein)

Question 26

Why do capping and tailing occur

Answer 26

Capping and tailing are thought to occur to facilitate export, confer stability, and facilitate the ribosome binding in cytoplasm.

Question 27

Describe alternative splicing

Answer 27

Sometimes introns may not be spliced out (or exons may still be spliced out - not 100%) and therefore alternative splicing can occur; a process by which different combinations of exons are joined together. This results in the production of multiple forms of mRNA form a single pre-mRNA/allows for multiple gene products from the same gene (we have ~20,000 genes, but there could be many times that number of proteins)

Question 28

How many genes do we have and do they vary in size

Answer 28

The human genome consists of roughly 3,000 million bp (haploid) with ~20,000 gene - but more proteins than that

There is big variation in gene and protein size.
For example, the TATA-box binding protein spans 18,000 bp in chromosome 6, has 8 exons and the final protein is 339 amino acids (average size). Comparatively, the Huntingtin gene spans 180,000 bp of chromosome 4, has 67 exons, and the protein has 3144 amino acids

Question 29

What happens after RNA processing

Answer 29

After this process the mRNA will leave the nucleus via a cell pore. There are specific proteins which determine what goes in and out of the nucleus (e.g., mRNA)

Question 30

What are the steps of translation and where does it occur

Answer 30

Initiation
Elongation
Termination

All translations start in free ribosomes, and are then taken to the ER if applicable (e.g. not a cytosol protein). If so, the protein will be put into the lumen of the rough ER, then be transported to Golgi via vesicle and transported again to do whatever their function is.

If this is the case:
A signal recognition peptide (STP) will attach to the signal peptide that at the start/N terminus of the POLYPEPTIDE CHAIN and will stop the process. Eventually the SRP will attach the ribosome to the RER and translation will continue. Once the chain is formed, a signal cleaving enzyme will cut off the signal peptide and the components disassemble. Importantly, the proteins will be produced and fold IN the ER (if applicable - as folding conditions are important), unless it is a membrane protein in which case it will stay anchored to the membrane - will then go to Golgi for further maturation

Question 31

Describe initiation (translation)

Answer 31

Small ribosomal subunit with initiator tRNA (tRNA carrying methionine/Met) already bound, binds 5’ cap of mRNA. Small ribosomal subunits scan downstream to find translation start site (AUG for eukaryotes). Hydrogen bonds form between initiator anticodon and mRNA. Large ribosomal subunit then binds, completing the initiation complex. Energy (GTP - guanosine triphosphate) is required for assembly.

Many additional initiation factors are required

Question 32

Describe elongation (translation) - three steps

Answer 32

Coding recognition: Weak hydrogen bonds form between mRNA base pairs and new complementary anticodon (tRNA with anticodons arrive and bind partially by chance as they will just be in the cytoplasm ready). GTP invested to increase accuracy/efficiency.

Peptide bond formation – The large subunit rRNA catalyses strong peptide bond formation (between amino acids). Then, bonds between the amino acid and the tRNA in P the site are broken.

Translocation - The tRNA in the A site will move to the P site while the tRNA in P site moves to E and is released. Energy is required (GTP).
Process continues

Empty tRNA are reloaded in the cytoplasm using aminoacyl-tRNA synthetases.

Additional elongation factors are required

REMEMBER THAT PROTEINS MAY BE MADE IN/ON THE RER

Question 33

How to empty tRNA get reloaded?

Answer 33

Empty tRNA are reloaded in the cytoplasm using aminoacyl-tRNA synthetases. Can therefore be reused

Question 34

Describe termination

Answer 34

Ribosome reaches a stop codon on mRNA and the mRNA stop codon in the A site is bound by a release factor.
Release factor promotes hydrolysis - bond between p-site tRNA and last amino acid is hydrolysed, releasing polypeptide.
Ribosomal subunits and other components dissociate. Hydrolysis of two GTP molecules is required (2GTP -> 2GSP + 2Pi). Ribosome components can be recycled.

Question 35

What are the ribosome binding sites for mRNA and tRNA

Answer 35

mRNA binding site - site on the small subunit where mRNA binds
A site (aminoacyl-tRNA binding site)- holds the tRNA that is ‘next in line’
P site (peptidyl-tRNA binding site) - holds tRNA carrying the growing polypeptide
E site (exit site) - tRNA exit from here

Question 36

Describe tRNA

Answer 36

The physical link between the mRNA and the amino acid sequence of proteins

Have an anticodon that bonds to a codon

Can be shown as a plane stem or loop model, ribbon model, or complete cartoons

Question 37

How are ribosomes assembled

Answer 37

Ribosomes (each subunit) are assembled in nucleolus and come out through nuclear pores and float around as separate subunits in cytosol.

The subunits will join once initiator tRNA and mRNA have bound and tRNA is bound to start codon

Question 38

What are the differences between mRNA and DNA

Answer 38

Key difference between DNA and RNA is that mRNA is single stranded and therefore less stable, we want DNA to be stable as it is there for the whole life of cell
mRNA has U not T

Question 39

Why is control of gene expression important

Answer 39

For most genes/proteins, you don’t want genes to be expressed all the time (proteins are only produced in response to stimuli as required)

With this said some do need to be produced constantly e.g. tubulin - therefore makes constantly/constant turnover and has a longer half-life
Housekeeping proteins - Proteins that are constantly produced

Other proteins are produced in response to stimuli as required:
- cell signalling
- signal transduced and may enter nucleus to activate transcription
- production of a short-lived protein to carry out the required function

Question 40

What are housekeeping proteins

Answer 40

Proteins that are constantly produced (e.g. tubulin)
- protein and mRNA are present in large quantities
- typically have longer half life in cells

Question 41

What are the control factors of gene expression and what is the strongest?

Answer 41

Transcription (the strongest): DNA needs to be accessible (methylation status - is it too condensed) and transcriptions factors need to assemble

Capping, extent of polyadenylation/tailing, alternative splicing, product an mRNA able to be translated

Specific proteins assist in nuclear export of mRNA

Regulatory proteins can block translation and variable mRNA life-spans

Question 42

Name and describe the protein structure types

Answer 42

Protein sequence determines its final structure which determines its function

Primary structure is just the polypeptide chain (amino acids bonded with peptide bonds - covalent bonds), doesn’t like to stay in this structure (C’ terminus end is made first and N’ terminus end is made last).

Secondary structures will form as they come out of the ribosome, where the skeleton of amino acids from the same protein are held together by hydrogen bonds. Two types, alpha helix and beta pleated sheet

Tertiary structure is a long peptide chain with some 3D shape but also folded together (many different but weak bonds involved - so boiling proteins typically breaks it). Bonds are between side chains of amino acids. For example, bonds such as phosphodiester bonds and disulphide bonds can form between side chains of amino acids on same protein to form tertiary structure

Quaternary structure - multiple polypeptide chains/proteins associate together to form a function protein, e.g., between ions (Ca2+)

ALL PROTEINS GO TO AT LEAST TERTIARY STRUCTURE AS THIS IS WHEN THEY FUNCTION, but not all go to quaternary

Question 43

What are post translational modifications

Answer 43

Modifications to proteins after folding - proteins may not yet be functional
These can confer activity (e.g. phosphorylation or enzyme cleavage)
Or impact ability to interact with other molecules (e.g. Biotinylation, methylation of histones)
Or direct to particular locations (e.g. ubiquitination for proteasome degradation)

Examples:
Phosphorylation (addition)
Methylation (addition of methyl group)
Acetylation (addition of an acetyl group)
Biotinylation (biotin added)
Carboxylation (carboxylic acid group added)
Carbohydrate addition (especially membrane bound proteins e.g. glycoproteins)
Cleavage
Ubiquitination

Some occur within the GOLGI others in CYTOSOL - especially if protein stays in cytosol (phosphorylation)

Question 44

Describe the basics of amino acids

Answer 44

There are 20 different amino acids.

Can have multiple codons for a single amino acid.

Ultimately made from amino group, carboxyl group, and a side chain

Amino - n end (will be on the N’ terminus of protein)
Carboxyl - c end (will be on C’ terminus of protein)

Side chains dictates the propertied of each amino acids and therefore what the proteins can interact with

Amino acids collectively determine the final structure and function of the protein

Question 45

Why do cells differ

Answer 45

Cells differ because different cells get signalled to do different things/express different genes - they all contain the same DNA but are clearly different