Lectures 8,9 and 10 - DNA, RNA and Protein synthesis

What did Mendels experiments lead to the idea of

Mendel studied pea plants and observed how traits were inherited. He discovered predictable ratios in trait inheritance, leading to the concepts of

• Genes as discrete units of inheritance

• Dominant and recessive traits

How did Miescher’s experiments describe evidence

provided the first chemical evidence of DNA by isolating it from cell nuclei. His discovery showed there was a unique chemical in the nucleus that could be involved in inheritance

How did Morgans experiments describe evidence

Morgan studied fruit flies and Morgan concluded that the gene for eye color was located on the X chromosome, which was the first solid evidence that genes are located on chromosomes which are apart of DNA

How did Griffiths experiments carry evidence

Used Streptococcus pneumoniae bacteria in mice. Found that a "transforming principle" from dead virulent (smooth) bacteria could turn non-virulent (rough) bacteria into virulent ones. This hinted that some heritable material was being transferred.

How did Chargraffs experiment describe evidence

His findings showed that DNA composition varied between species but same within species, but the A=T and G=C pairing is constant—key evidence that helped Watson and Crick later deduce the double helix structure of DNA.

How did Franklins experiment describe evidence

Rosalind Franklin used X-ray crystallography to capture images of DNA, revealing its helical structure

How did Watson and Crick describe genetic evidence

Watson and Crick came up with the DNA structure from Franklin to build a model of DNA’s double helix as it allowed to deduce the width of the helix and the spacing of the nitrogenous bases, showing how genetic information is stored and copied through complementary base pairing.

what kind of information did Watson and Crick come up with

• 2 hydrogen bonds between A and T

• 3 between G and C

• 10 base pairs a turn

• strands run antiparallel

• bases are non polar, hydrophobic

• the phosphate is polar, hydrophilic

what bases are purines and what does it mean

adenine and guanine - 2 fused rings

what bases are pyrimidines and what does it mean

thymine and cytosine - 1 fused ring

what makes DNA anti parallel

the two strands of DNA run in opposite directions, the leading strand runs from 5’ to 3’ whereas the lagging strand runs from 3’ to 5’

what allows the dna to twist into a double helix

A and T form 2 hydrogen bonds and G and C form 3 hydrogen bonds and this holds the dna strands in a stable manner and allows them to twist into a double helix

what does the anti parallel structure ensure

that the bases line up correctly

Conservative model for dna replication

the parental DNA molecule stays completely intact, and a new DNA molecule is synthesized. After one round of replication, there is one molecule with both old strands and one with both new strands. After the second round, there is one fully old DNA molecule and three fully new ones.

what is the semi conservative model

after the first round of replication, both DNA molecules consist of one old strand and one new strand. After the second round, there are two mixed DNA molecules (one old + one new strand) and two fully new DNA molecules (both strands newly made)

what is the dispersive model

each daughter strand contains strands made of new and old synthesized DNA

how did their experiment show that dna is conservative

The Meselson-Stahl experiment proved DNA replicates semi-conservatively.

• used isotopes of nitrogen to label DNA

• saw each new DNA molecule had one old strand and one new strand.

• the results were consistent with the semi conservative model.

• density gradient centrifugation was used to separate the two types

Role of helicase in DNA replication

unwinds the parental helix at replication forks

role of single stranded binding protein

binds to and stabilises single stranded DNA until it can be used as a template

role of topoisomerase

corrects overwinding ahead of replication forks by breaking, swivelling and rejoining DNA strands

role of primase

synthesizes a single RNA primer at the 5’ end of leading strand and at the end of each Okazaki fragments on lagging strand

role of dna polymerase 2

continuously synthesises the leading strand, adding on to the primer, elongates each Okazaki fragments on the lagging strand

role of dna polymerase 1

removes primer from 5’ end of leading strand and replaces it with DNA, adding on to the adjacent 3’ end, does the same on lagging strand but for each fragment

role of dna ligase

joins the 3’ end of the DNA that replaces the primer to the rest of the leading strand, joins Okazaki fragments in lagging strand

what is the error rate of dna replication

1 in 10^9 base pairs

what reduces the error of dna replication

the specific base pairing reduces the error rate and dna polymerases have proofreading ability and can detect and remove incorrect bases

what can rare mistakes lead to in dna replication

mutations which can cause defects in proteins (can be single nucleotide substitution leading to missense, nonsense or silent with no effect, can be insertion or deletion of nucleotides such as frameshift mutations, insertions and deletion)

how can dna be repaired

DNA can repair mutations caused by radiation or chemicals through a process where a thymine dimer distorts the DNA molecule. A nuclease enzyme will cut out and remove the mutated section, DNA polymerase fills in the missing nucleotides, and DNA ligase seals the strands together, restoring the DNA.

DNA replication

1. Replication starts at specific origins of replication (one in prokaryotes and one in eukaryotes).

2. Proteins recognise the DNA sequence and bind to it, opening up the double helix. In the replication bubble there are Y shaped replication forks where the new strands are being elongated

3. Replication starts at multiple sites where the parental strands separate to form replication bubbles

4. The bubbles expand laterally as DNA replication proceeds in both directions

5. Eventually the replication bubbles fuse and synthesis of the daughter strands are complete

what is the elongation if dna replication carried out by

enzymes known as dna polymerases, they use triphosphates (ATP, GTP) as the sources of nucleotide units to add to the growing chains. A 2 phosphate unit (pyrophosphate) is split out as the chain is extended by each nucleotide

what is the rate of elongation in a second in humans

50 nucleotides

why is there a leading and a lagging strand

because the strands are anti parallel and the enzymes can extend chains in one direction only, 2 strands cannot be replicated continuously

how long are the okazaki fragments

100-200 nucleotides in length

How does the process of dna polymerases extending a pre existing strand start

the cell starts by making a short primer of RNA about 10 nucleotides long

hi

b3 for the win

what is mitochondrial DNA and what do the mutations usually cause

mtDNA is passed through generations via maternal lineage, mutations usually cause the inability to generate through energy

Describe the evidence for the link between genes and proteins as shown by studies of mutants

Garrod (1909) studied patterns of inherited disease such as albinism and alkaptonuria, coined the term ‘inborn errors of metabolism’ linked symptoms of an inherited disease with a persons inability to make a particular protein

- Beadle and Tatum (1930s-40s) studied link in detail using mutants bread mould (Neurospora crassa),they studied mutations in pathway in which arginine is synthesised. they set up three classes with a defective gene in each, each mutant was unable to carry out one step in the pathway for arginine synthesis = genes do not build protein, the link is indirect, mrna is the intermediate

does rna polymerase have proof reading capabilty

no error rate is 1 in 10^4

how does rna polymerase copy dna into rna during transcription

by using one dna strand as a template. RNA polymerase binds to DNA at the promoter region and unwinds the double helix. It then reads the template strand in 3’ to 5’ direction and synthesises the complementary strand in 5’ to 3’ direction. The DNA template provides the sequence of the bases for the mRNA transcript. Once the termination signal is reached (AAUAAA) polyadenylation signal, the RNA is released

How does RNA polymerase find the start of the gene and make the transcript of the template strand of DNA in the correct direction

RNA polymerase recognises the promoter near the beginning of the gene where it binds. In eukaryotes the promoter often contains the TATA box and a set of transcription factors are involved in binding RNA polymerase II to the promoter. In prokaryotes it contains the -10 and -35 regions recognised by the sigma factor of RNA polymerase.

Once the strand is unwinded, RNA polymerase starts transcription. The template strand runs in the 3’ to 5’ direction so the copy is made from 5’ to 3’.

how are RNA transcripts in eukaryotic cells processed into their mature functional forms and transported to their correct location in the cell

In eukaryotic cells

- A cap is added to the 5’ end (modified form of G)

- A polyA tail is added to the 3’ end (50-200 nucleotides long)

They protect mRNA from degradation, aid export from nucleus and help mRNA to anchor to ribosomes.

what happens after capping and tailing

the mrna is spliced to remove non coding regions (introns) and leaves the exons (coding regions)

what protein carries out the splicing and what are they

small nuclear ribonuclearprotein, consist of small nuclear RNA (150 nucleotides long) and various proteins, also play a catalytic and structural role aswell

what do several snRNPS form

a spliceosome

how does elongation proceed

by local unwinding of 1-2 turns of the dna and addition of nucleotide units (about 60 per second)

explain that cells need to make polypeptides of defined length and sequence

Cells need to make polypeptides of defined length and sequence because the specific order of amino acids in a polypeptide determines its structure and function. A correct sequence ensures that the polypeptide folds properly into a functional protein, which is essential for carrying out specific cellular tasks. if the sequence or length is incorrect, the polypeptide may be harmful or nonfunctional to the cell

Describe the importance of base-pairing in ensuring the accuracy of protein synthesis

base pairing ensures that the mRNA sequence accurately reflects the gene encoded in the DNA.

Base pairing with the codons of the mRNA and anticodons on tRNA ensures that the correct amino acids are brought to the ribosome to add to the polypeptide chain in order to make the correct protein.

Describe the machinery (ribosome)

The ribosome is made up of rRNA (65%) and ribosomal proteins. It has a large subunit (50S) and a smaller subunit (30S) which join together when mRNA is present.

It has some key binding sites for translation:

- 30 S a site for binding mRNA (30S)

- 50 S A site (aminoacyl-tRNA) where the incoming aminoacyl-tRNAs bind

- 50 S P site (peptidyl-rRNA) where the growing polypeptide chain binds

- 50 S E site where discharged tRNAs exit the ribosome

Describe the raw materials (aminoacyl-trnas)

The loading of trna with its correct amino acid is carried out by aminoacyl-trna synthetase, Thos joining reaction requires ATP, there are 20 such enzymes and each one has an active site that houses a specific combination of trna and amino acid

Describe the instruction tape (mRNA)

carries genetic code from DNA to the ribosome for protein synthesis. The sequence of nucleotides in mRNA is read in triplets of bases (codons). Translation starts at the start codon (AUG – methionine) in 5’ to 3’ direction. The process stops at the stop codon (UAA, UAG or UGA).

describe protein synthesis (translation)

Initiation: The 30S ribosome subunit binds to the mRNA at the start codon (AUG), guided by the mRNA sequence (prokaryotes) or the 5' cap (eukaryotes). A special initiator tRNA carrying methionine binds to the start codon. The 50S subunit binds, forming the initiation complex, with initiation factors and GTP required. The first tRNA enters the P site.

Elongation: Amino acids are added one by one in three steps:

1. Codon recognition: The correct aminoacyl-tRNA enters the A site, requiring energy (2xGTP) for accuracy and efficiency

2. Peptide bond formation: The newly arrived amino acid is joined to the growing peptide chain anchored in the A site, catalysed by rRNA in the large subunit.

3. Translocation: The ribosome moves the tRNA from A to P site, and the discharged tRNA moves to the E site and exits, requiring energy (GTP).

This cycle repeats, adding 10-20 amino acids per second as the ribosome moves 5' to 3' along the mRNA.

Termination: When a stop codon (UAA, UAG, or UGA) is reached, a release factor protein binds instead of tRNA, and the completed peptide chain is released. the components of the system come apart and can be used for another translation cycle

Explain the main characteristics of the genetic code which defines the association of codons with anti-codons

Triplet nature: 3 nucleotides code for 1 amino acid. Non-overlapping code that must be read in the correct reading frame. It is degenerate, most AAs are coded by one or more codon, known as synonyms, to act as a buffer against mutations. Unambiguous as each codon specifies only one AA or stop signal.

Explain that, for proper cell function, proteins must be processed and targeted to their correct cellular locations

After leaving the ribosome, a number of processes occur:

- Protein folding into 3D structure required for function

- Post-translational modification: phosphorylation, acetylation etc.

- Cleavage : some proteins must be cleaved to become active

Protein targeting

New proteins destined for export from the cell or membrane have short signal sequences on the N-terminal which direct them to the correct location.

Also, specific transport mechanisms help move the protein to its destination such as nuclear pores, membrane channels or vesicles.