Maintaining the Cell and Genome

Question 1

What is a genome?

Answer 1

Complete set of genetic information in an organism

Provides all the information an organism requires to function

Genome is stored in long molecules of DNA: chromosomes

In eukaryotic cells, the genome is contained within the nucleus

Question 2

How is DNA stored/organised in a cell?

Answer 2

DNA packaged with histones (positive charged) – form nucleosome structure – nucleosome are the functional unit of chromatin – fold into a tightly coiled structure - ultimately condenses into chromosomes

Question 3

What are the building blocks that make up DNA? What are they composed of?

Answer 3

The basic building block of DNA is the nucleotide

1. 5-carbon sugar
2. Phosphate moiety
3. Nitrogenous base - nitrogen containing ring

Things to note…
1. Sugar-phosphate backbone gives the DNA strand polarity - 5’ carbon of one sugar binds to the 3’ carbon of the next via a phosphodiester bond
2. Four different types of bases - A, T, C and G

Question 4

What are the two groups of nitrogenous bases?

Answer 4

Note:
- Purines are bulkier as they have a double ring structure

Question 5

What type of bonding holds two DNA strands together?

Answer 5

A-T – two hydorgen bonds
C-G – three hydrogen bonds

Question 6

What is the difference between a nucleotide and a nucleoside?

Answer 6

Question 7

Do DNA strands have directionality?

Answer 7

YEAH BUDDY

Question 8

How are nucleosomes formed?

Answer 8

First and most fundamental ‘level’ of chromatin packing is the nucleosome:

Histones + DNA – nucleosome 200 Bp – come together to form ‘beads on a string’ chromatin

Question 9

What are the subunits that make up histones? What other important features do histones have?

Answer 9

Histones – DNA-binding proteins (small positively charged proteins).
Two each of: H2A, H2B, H3 & H4 - forming an octamer

Histone tails are significant – susceptible to a large number of covalent modifications

Question 10

After nucleosome formation, what are the following levels of DNA condensation/packaging?

Answer 10

1. First order packing – beads on a strings – 7 fold reduction in length
2. Chromtain fibres - looping/organising histones
3. Chromtain fibres undergo further looping
4. Formation of mitotic chromosome

Question 11

How many chromosome, autosomes and sex chromosomes do we have?

Answer 11

23 chromosomes - Haploid Cell
46 chromosomes - Diploid cells = 3 billion nucleotide (base) pairs

22 pairs of autosomes
1 pair sex chromosomes

Question 12

What is meant by the idea that DNA replication is semi-conservative?

Answer 12

Due to WC base pairing - each strand acts as template for synthesis of a new complementary strand - Base pairing enables DNA replication

Hence, after replication the new DNA strand will carry a parent and a new daughter strand - hence, DNA replication is semi-conserved.

Question 13

Where does DNA replication begin? Where does DNA replication take place? What are these sites called?

Answer 13

DNA synthesis begins at replication origins

Replication origin – sites where DNA replication starts – need to seperate strands and create a replication bubble (break open hydrogen bonds) - creates single stranded templates ready for DNA synthesis

Initiator proteins recognize replication origin and unwind DNA at these sites

Multiple origin sites can be active at one time

DNA synthesis occurs at replication forks

Two forks form at each origin

Replication proceeds bidirectionally, unzipping the DNA strands as it goes

Most important enzyme in DNA replication is DNA polymerase

Question 14

In what direction is DNA synthesized?

Answer 14

DNA is synthesised in the 5’ to 3’ direction

Question 15

What big problem is encountered in DNA replication?

Answer 15

At a replication fork, the two newly synthesised DNA strands are of opposite polarities

This creates a problem for DNA polymerase, which can only synthesise DNA in a 5’-3’ direction

Question 16

How does DNA replication of the lagging strand take place?

Answer 16

Solution – synthesizes fragments on the lagging strand in a 5’ to 3’ (work in the opposite direction to the replication fork) forming Okazaki fragments

These can then be ligated together

This is known as discontinuous synthesis

Question 17

Why are primers needed for DNA replication? What enzyme is responsible for laying primers down?

Answer 17

Problem - DNA polymerase can only continue an existing strand, not initiate new ones - hence, this is why primers are needed

An RNA polymerase known as primase makes RNA primer first (~10 bases long) - DNA polymerase can then extend the RNA chain

Note
- Only one RNA primer needed for leading strand, but lagging strand has continuous requirement
- RNA later removed by nuclease activity

Question 18

Does DNA polymerase proof-read its own work?

Answer 18

Yes, DNA polymerase proofreads its own work

Replication must be very, very accurate (although mutations must arise at some frequency, or evolution couldn’t occur)

Consequences of errors can be fatal

Allowing for proofreading, DNA polymerase makes around 1 error in 10^7 bases

Question 19

What is the overall accuracy of DNA replication? What mechanism exists to correct these mistakes?

Answer 19

Overall accuracy of DNA replication is ~1 error in 10^9 bases

Mutations can be passed on to offspring if they take place in germline cells but can also be problematic in somatic cells

DNA mismatch repair
Machinery recognizes error and new daughter strand – chews back and allows DNA polymerase to jump back in to repair

Question 20

What are causes of DNA damage?

Answer 20

Question 21

What is an example of DNA damage associated with the base thymine?

Answer 21

Exposure to ultraviolet light can form thymine dimers

If they can’t be repaired - results in a condition called Xeroderma pigmentosum (Genetic defect) - results in severe skin lesions, including skin cancer

Question 22

What is the principle behind DNA repair pathways?

Answer 22

Question 23

What are the differences between mitochondrial and nuclear DNA?

Answer 23

Question 24

What is the central dogma of molecular biology?

Answer 24

Question 25

What are the differences between DNA and RNA?

Answer 25

Extra DNA – mainly used as information storage RNA – plays a variety of roles – catalytic, make proteins, etc.

Question 26

Which carbon in the sugar moiety of DNA and RNA is different?

Answer 26

Carbon 2 RNA - Carbon 2 is hydroxylated DNA - Carbon 2 is not hydroxylated

Question 27

What is DNA transcription?

Answer 27

Transcription - Process that produces an RNA copy of the coding strand of DNA (except that it contains U, not T) Note - the non-coding strand is used as a template to form a coding strand

Question 28

What enzyme is responsible for transcription?

Answer 28

Transcription is carried out by RNA polymerase mRNA is made by RNA polymerase II - Makes an RNA copy of one DNA strand Recognizes start site - unwinds DNA - proceeds along DNA while transcribing new strand - new strand is released from complex - stop site reach - complex is released and DNA can re-anneal Requires 1. DNA template 2. Activated precursors (nucleoside triphosphates - ATP, GTP, UTP and CTP) 3. Does not require a primer 4. Synthesises in 5’- 3’ direction Note - Error rate is higher – mistakes have less severe consequences than DNA mutations

Question 29

Describe what happens in the RNA polymerase complex during transcription.

Answer 29

1. DNA enters complex 2. DNA is unwound 3. Exposing the non-doing/template strand to incoming rNTPs 4. rNTPs are added to the growing RNA chain (short DNA-RNA hybrid) 5. RNA released from DNA 6. DNA is allowed to reanneal

Question 30

How do we get tissue/cell specific expression of genes?

Answer 30

Genes may be either on (expressed) or off in a given cell - not all genes are expressed in all tissues at all times Genes may be expressed in a: 1. Tissue-specific pattern 2. Developmentally regulated pattern 3. Combination of these 4. “on” all the time, in all tissues (constitutive) - e.g. housekeeping genes How? - Transcription factors interact with the 5' promoter and the enhancer sequences to regulate expression (increase or decrease)

Question 31

What is the transcription factor responsible for myosin gene expression?

Answer 31

Presence of transcription factors MyoD will dictate whether the myosin gene is transcribed or not

Question 32

How can a single 'gene' give rise to different protein products?

Answer 32

Expression of a single gene can be controlled at various levels to give different products: 1. **Alternative promoters** - different transcription start sites (TSS) used resulting in different products. 2. **Alternative splicing** - primary transcript can be spliced in different ways, joining different exons together, resulting in different products (not exon fundamental order can't be changed - they can simply be skipped) e.g. Example: the alpha-tropomyosin gene can be spliced in many different ways 3. **Alternative polyadenylation**

Question 33

Before exiting the nucleus, what modifications do mRNA transcript undergo?

Answer 33

Before RNA can be exported from the nucleus it must go through RNA processing steps: 1. Splicing 2. Capping 3. Polyadenylation

Question 34

What is RNA capping? Why is it important?

Answer 34

Modifies the 5’ end of a RNA transcript The RNA cap includes an atypical nucleotide: a guanine nucleotide that has a methyl group attached to the 5’ end of the RNA in an unusual way Important for... protecting RNA from degradation, stabilization, stimulates splicing and drives nuclear export

Question 35

What is polyadenylation? Why is it important?

Answer 35

A polyA tail is a string of adenylate residues added to the 3’ end of an mRNA Not found on rRNAs or tRNAs - Labels the RNA as mRNA Transcription proceeds past polyA site, transcript is cleaved, then polyadenylated

Question 36

What are 5' and 3' UTRs? Why are they important?

Answer 36

**5’ untranslated (5’UTR)** is the region of an mRNA that is found upstream of the translated region Function of 5’UTRs is mostly unclear, may affect translational control **3’ untranslated (3’UTR)** is the region of an mRNA that is found downstream of the translated region 3’UTRs can determine the stability of the mRNA

Question 37

What are the three major classes of RNA?

Answer 37

Question 38

What are some examples of non-coding RNAs and their functions?

Answer 38

Extra 1. Circular RNA species - Class of RNA molecules with closed loops, high stability, abundantly expressed in eukaryotic organisms - thought to regulate gene expression and protein translation

Question 39

What is PCR? What is it used for? What are the steps undertaken in this reaction?

Answer 39

The Polymerase Chain Reaction (PCR) - DNA amplification - can be used for genotype - detecting mutations in patients Steps 1. Strand Seperation 2. Primer annealing 3. Extension

Question 40

What happens during the process of translation?

Answer 40

Translating – change in ’code/language’ - change from nucleotides (DNA/RNA) into protein The RNA code is read in triplets (codon - three bases at a time) - with each triplet codon corresponding to a specific amino acid

Question 41

What is meant by the genetic code being redundant?

Answer 41

Redundancy in the genetic code - multiple codons coding for a single amino acid - triplets can code for 64 different combinations but there are only 20 amino acids Note - Third position in the codon is commonly redundant - There are also start (AUG) and stop codons (UAA, UGA and UAG)

Question 42

How are codons read? What are reading frames?

Answer 42

- Code is read in the 5’ to 3’ direction - Three different reading frames – depends where you start counting the triplets - Important that translation starts at the correct start position/reading frame

Question 43

What molecule is responsible for translating/bringing amino acids to the corresponding codon?

Answer 43

tRNAs (transfer RNAs) are key adaptors required for protein synthesis tRNA has a 3' end that attaches to a specific amino acid and a anti-codon region that binds to the codons on the mRNA strand - therefore allowing it to bring the corresponding correct amino acid Note - Some tRNAs require accurate base-pairing only at 1st two nucleotides - This ‘wobble’ accounts for finding that many alternative codons for a particular amino acid differ only in their 3rd nucleotide

Question 44

How is the correct amino acid added to the corresponding tRNA molecule?

Answer 44

**tRNA synthetases** are needed to transfer the correct amino acid to the corresponding tRNA Recognizes anti-codon and matches with the correct amino acid – relies on ATP hydrolysis to bind the two together (charging of the tRNA) – forms a high energy bond which is required for the addition of the amino acid to the growing peptide chain

Question 45

What are ribosomes? What role do they play in translation?

Answer 45

Ribosomes - the factory of translation - very complex - Made of RNA (rRNAs) and proteins - Has large & small subunits - Large subunit – catalyzes the peptide bond formation – covalently linking amino acids - Small subunit – matches tRNA to mRNA - Ribosome moves along the mRNA transcript, in a 5’ to 3’ direction Role - guides translation and catalyzes the polymerisation reaction

Question 46

What are the three tRNA docking sites on ribosomes?

Answer 46

How does it coordinate this reaction Three tRNA sites and mRNA binding sites 1. A site – aminoacyl site - tRNA binding 2. P site – Peptidyl site - peptide bond formation/polymerisatrion 3. E Site – exit

Question 47

Outline the steps that take place in the translation cycle in order to produce a polypeptide chain.

Answer 47

1. tRNA for start codon binds to the P site directly (bringing in Methionine) 2. tRNA with the matching anti-codon for the codon in the A site binds 3. Peptide bond is formed between the amino acid in the P and A site – causes release of the amino acid from it’s tRNA 4. Ribosome shifts across - Large subunit translocates, followed by small subunit translocation 5. Spent tRNA in the E site leaves 6. Vacant A site can be filled by new tRNA molecule 7. Process repeats.

Question 48

How/where is translation started and stopped?

Answer 48

1. Translation initiates at a methionine codon (AUG) near 5’ end of mRNA. Therefore, methionine is always the first amino acid added (may be removed later) 2. Translation terminates at a stop codon. Three stop codons, UAA, UAG and UGA - Stop codons not recognised by a tRNA, release factor binds instead

Question 49

Do some antibiotics target prokaryotic translation?

Answer 49

Yes, some antibiotics work by inhibiting prokaryotic protein synthesis This is possible as there are differences in the structures of prokaryotic and eukaryotic ribosomes