DNA, RNA and gene expression

Purine

A type of nitrogenous base with a double-ring structure, which includes adenine (A) and guanine (G).

Pyrimidine

A type of nitrogenous base with a single-ring structure, which includes cytosine (C) and thymine (T).

Helicase

An enzyme that unwinds the double-helix structure of DNA during the replication process.

Ligase

An enzyme that joins the ends of DNA strands by forming new phosphate bonds.

Exonuclease

An enzyme that removes nucleotides from the ends of a DNA strand, functioning in both 3’ to 5’ and 5’ to 3’ directions.

Origin of replication

The specific location on the DNA molecule where replication begins.

In EUKARYOTES there are multiple origins replication.

Bidirectional in Eukaryotes

Bases added at a rate of 1000/second

So replication of the whole mammalian genome takes approximately 8 hours

In PROKARYOTES there is only ONE origin of replication

Telomere

The repetitive DNA sequences located at the ends of linear chromosomes, important for maintaining chromosome integrity.

G:C rich repeats

In humans  - TTAGGG/AATCCC  - highly conserved

NON-CODING but ESSENTIAL to maintain the integrity of DNA

Nucleoside

sugar + base

Phosphate attaches to 5' carbon

1st Carbon binds to base through N-glycosidic bond = stability to sugar and base (nucleoside)

Anti-parallel

Bonding between bases

Different H-bonds between bases allows a twist to the antiparallel structure = DNA double helix. Compacted in such a way, it can be unravelled for eukaryotic processes

Major/minor groove

Sequences that are accessible on outside of major groove allows transcription factors or replication machinery to actually bind into the minor or major groove

Phosphate on outside of two antiparallel strands and bases are in middle = helix can form

5’ to 3’

5' = phosphate group

3' = free hydroxyl group

Asexual reproduction - prokaryotes

1. The entire genome is on one circular chromosome = DNA molecule

2. The chromosome replicates once to produce two chromosomes that are identical (except for rare mutations).

3. The two identical daughter chromosomes move toward opposite end of the cell.

4. When the cell divides the daughter chromosomes are partitioned one to each daughter cell.

Asexual reproduction - eukaryotes

DNA replicates during S

Gene expression occurs during G1 and G2 (and S?)

Nuclear division (mitosis) occurs during Mitosis

Cell division (cytokinesis) occurs at the end of Mitosis

Some genes may be expressed in S phase but they will be genes that aren't highly expressed

Highly expressed genes will be replicated early in S phase - replication and transcription can't happen simultaneously

Silent genes are replicated late so there can be some transcription

General features of DNA replication

•semi conservative

•It is bidirectional process

•It proceed from a specific point called origin

•It proceed in 5’-3’ direction

•It occur with high degree of fidelity

•It is a multi-enzymatic process

occurs by three steps

1. Initiation

2. Elongation

3. Termination

Single-strand binding protein

binds to and stabilizes the single strand to keep DNA unwound.

bind on either strand

Primase

adds ribonucleoside triphosphates to synthesise an RNA primer

Binds at the initiation point of the 3'-5' parent chain

Bind to 3' to 5' parent strand because primase runs in 5' to 3' direction to lay down the primer

DNA polymerase

proceeds in a 5’ to 3’ direction

Adds 1000 bases /second to the growing chain

Requires all 4 dNTPs deoxyribonucleotides

MUST have a template and a primer

Has proof reading activity - Will shuffle back and check its laid down the right bases = fidelity

attach to RNA primer

Initiation

Primase lays down a primer

Single strand binding proteins are going to bind to each of the parent strands

In a 5' to 3' direction on both strands and therefore using 3' to 5' parental strand as template

elongation

DNA polymerase extends in a 5’ to 3’ direction

DNA polymerase attaches to RNA primer and then lays down comp. bases according to parent strand in 5' to 3' direction on both strands

Exonuclease removes the RNA primer

Polymerase fills the gap

Ligase joins the two pieces ia a condensation reaction forming 3' to 5' phosphodiester bonds

Can remove a primer because it is RNA and therefore has a different sugar and bases

Okazaki fragments

The lagging strand has to wait for the helicase to break the H-bonds before it can lay down an RNA primer for the DNA polymerase to replicate. Therefore it's made up of many small replicated DNA fragments called Okazaki fragments

Hydrogen bonding is still occurring when the DNA polymerase moves from a 5' to 3' direction behind the helicase that is breaking the H-bonds - this is the leading strand because its able to continually replicate because the helix is in front of it

Termination

It takes 8 hours for DNA to be replicated

At the end of DNA replication the RNA primer are replaced by DNA by 5’-3’ exonuclease and polymerase activity of ε DNA polymerase.

Exonuclease activity of DNA polymerase removes the RNA primer and polymerase activity adds dNTPs at 3’-OH end preceding the primer.

In eukaryotic organism with linear DNA, there is a problem.

When RNA primer at 5’ end of daughter strand is removed, there is not a preceding 3’-OH such that the DNA polymerase can use it to replace by DNA. So, at 5’ end of each daughter strand there is a gap (missing DNA). This missing DNA cause loss of information contain in that region. This gap must be filled before next round of replication.

Enzymes that finish replication

1. Gyrase (a topoisomerase)

2. telomerase

Gyrase (a topoisomerase)

relaxes supercoils produced when the molecule is twisted during replication. Also facilitates unwinding at beginning of replication.

Telomerase

RNA-dependent DNA polymerase

uses a short RNA template to add short DNA repeats to the short ends of linear chromosomes when the last primer is removed using RNA template

Telomeres get shorter by 10 to 15 bases each replication so older people are at a higher risk of age-related diseases due to shorter telomeres

regulatory proteins

don’t code but regulate expression and stability

RNA

Ribose has a hydroxyl group on 2nd carbon instead of hydrogen like DNA

Hydroxyl group makes more prone to degradation because phosphodiester bond is less stable than in DNA

DNA is very stable whilst RNA is not partially due to differences in sugars

RNA composition

A, G, C, U

Uracil - Demethylated thymine = less stable

Less resistant to photochemical mutations

properties of RNA

single-stranded - 5' end up into a loop down to 3' end. The stem will have comp pairing between bases that aid stability and allow RNA to remain in nucleus for enough time. Allows RNA to be less vulnerable to degradation

Read from 5' to 3'

RNA sequence comes from its DNA

Carries genetic info

Always shorter than DNA in eukaryotes

Can form hairpin structures so that it is stable for enough time for the protein to be expressed

Gene codes, RNA expresses

tRNA

Transfer RNA: Brings amino acids to ribosomes during translation.

4 loops - conserved 3 loops and free loop

are encoded by tRNA genes.

All tRNA molecules are similar in size and shape.

have CCA at the 3' end to which the amino acid attaches

At the other end is the ANTICODON, which, during translation, "reads" the matching codon on the mRNA.

mRNA

Messenger RNA: Encodes amino acid sequence of a polypeptide.

Single RNA strand

4 domains- size depends on transcript length of gene

Transcription summary

1. DNA unravels to be accessible to transcriptional machinery

2. Helicase and topoisomerase keeps DNA in open confrontation for transcription

3. RNA polymerase read code and make comp template pre-mRNA using other stand as template

4. Pre-mRNA - transcript of whole coding region

5. Pre-mRNA is processed via RNA processing = shorter mRNA

6. All of this is in nucleus

7. mRNA exported out of nuclear pore into cytoplasm. Then trafficked to RER where ribosomes are and is translated

8. Whole processes also in mitochondria for mitochondrial DNA

4 types of RNA

If infidelity in any RNA form can lead to mutation with detrimental effect  due to them being fundamental genes within eukaryotic cell

1. mRNA

2. tRNA

3. rRNA

4. snRNA

rRNA

Ribosomal RNA: With ribosomal proteins, makes up the ribosomes, the organelles that translate the mRNA.

snRNA

Small nuclear RNA: With proteins, forms complexes that are used in RNA processing in eukaryotes. (NOT found in prokaryotes.)

A protein-coding gene

consists of a PROMOTER followed by the CODING SEQUENCE for the protein and then a TERMINATOR.

promoter

a base-pair sequence that specifies where transcription begins

at 5' end

Variable in sequence with some highly-conserved elements

Has transcriptional start site - allows transcription factors and RNA polymerase to bind

coding sequence

a base- pair sequence that includes coding information for the polypeptide chain specified by the gene.

Mutations due to errors in DNA replication or DNA exposure to mutagens can have detrimental or advantageous effects if within crucial regions like promoter or coding sequence

terminator

a sequence that specifies the end of the mRNA transcript

Transcriptional end site

TRANSCRIPTION- BIOSYNTHESIS OF mRNA

RNA Polymerase recognises the PROMOTER and begins TRANSCRIPTION Pre-mRNA is synthesised in a 5’ to 3’ direction

As the Polymerase moves along the DNA template the RNA is released and the DNA helix reforms

Once the Polymerase hits the terminator sequence it STOPS and the Full Pre-mRNA transcript is released.

In order for RNA polymerase to work in a 5' to 3' direction, it must use the parent 3' to 5' strand as its template

coding strand

not used as a template, but is identical in sequence to the mRNA except that all the U's are T's

template strand

what is used as a template in the synthesis of mRNA

mRNA in Prokaryotes

The sequence of a prokaryotic protein-coding gene is colinear with the translated mRNA.
The transcript of the gene is the molecule that is translated into the polypeptide.

No RNA processing happens - it has the same structure of the gene it has transcripted

mRNA in Eukaryotes

The sequence of a eukaryotic protein-coding gene is typically NOT COLINEAR with the translated mRNA.

The transcript of the gene is a molecule that must be PROCESSED to REMOVE these extra sequences called ‘INTRONS’ BEFORE it is translated into the polypeptide.

RNA processing - adds additional stability to RNA. It is processed

Both ends of a eukaryotic pre-mRNA molecule are modified by enzymes during transcription, and these modifications remain in the mRNA that is produced

At the 5' end, a cap is added consisting of a modified GTP (guanosine triphosphate). This occurs at the beginning of transcription. The 5' cap is used as a recognition signal for ribosomes to bind to the mRNA.

At the 3' end, a poly(A) tail of 150 or more adenine nucleotides is added. The tail plays a role in the stability of the mRNA - Allows it to be within the nucleus and cytoplasm for longer

pre-mRNA

Most eukaryotic protein-coding genes contain segments called introns, which break up the amino acid coding sequence into segments called exons. The transcript of these genes is the pre-mRNA (precursor-mRNA).

The pre-mRNA is processed in the nucleus to remove the introns and splice the exons together into a translatable mRNA.

Pre-mRNA Processing (Splicing)

1. The intron loops out as snRNPs (small nuclear ribonucleoprotein particles, complexes of snRNAs and proteins) bind to form the SPLICESOME

2. The intron is excised, and the exons are then spliced together.

3. The resulting mature mRNA may then exit the nucleus and be translated in the cytoplasm.

Lysine

Highly positively charged A.A and important for condensing DNA information into a structure within the nucleus that allows it to be compacted and also allows it to be opened up when it is require to be replicated or transcribed

Start codon

AUG = Methionine

stop codon

UAA, UAG. UGA

degenerate

In most cases more than one codon per amino acid (max. 6)

Partial DEGENERACY - first two nucleotides are identical but the third (i.e., 3′ base) nucleotide of the degenerate codon differs

complete DEGENERACY - any of the 4 bases can take third position and still code for the same amino acid

How do we READ the RNA and make a peptide

1) Once a gene has been sequenced we need to identify the OPEN READING frame

2) Every region of DNA has SIX possible reading frames (three in each direction)

BUT remember DNA is double stranded

ATG when transcribed = Aug = MET = start codon

Have to write N-Met to show start

C has to be written to show stopping

read from 5' to 3' at all times

open reading frame (ORF)

run of codons that starts with ATG and ends with a termination codon, TGA, TAA or TAG.

promoter

regulatory region of DNA located upstream (towards the 5' region) of of a gene, providing a control point for regulated gene transcription

enhancer

short (50–1500 bp) region of DNA that can be bound by proteins –ACTIVATORS to > the transcription of a particular gene

TATA box

a sequence of DNA, consisting of nucleobases TATAAA, located in the promoter region about 25-30 base pairs before the site of transcription

Most promoter regions of genes do not contain a TATA box. In TATA-less genes, transcription factors recognize other promoter sequences and RNA polymerase binds to these instead.

motif

allows identification of important region for transcription. In genes that don’t have tata box

ribosomes

the organelles on which the mRNA is translated, consist of two subunits, each of which contains rRNA and ribosomal proteins.

large Subunit [60S] - 28S rRNA, 5.8S rRNA, 5S rRNA + ~50 ribosomal proteins

Small Subunit [40S]- 18S rRNA + ~ 30 ribosomal proteins

Adding an Amino Acid to tRNA

AMINOACYLATION or ‘CHARGING’- An enzyme called AMINOACYL-t RNA SYNTHETASE adds the correct amino acid to its tRNA.

Since there are 20 amino acids, there are 20 aminoacyl- tRNA synthetases.

All tRNAs with the same amino acid are charged by the same enzyme, even though the tRNA sequences, including anticodons, differ.

1. tRNAs are identified by their synthetases by contacts that recognize a small number of bases, typically from 1-5.

2. Often one of the last three base pairs in the acceptor stem is recognized. An extreme case is represented by alanine tRNA, which is identified by a single uniqu base pair in the acceptor stem.

3. Usually (but not always), at least one base of the anticodon is recognized. Sometimes all the positions of the anticodon are important.

   

Elongation of the Polypeptide Chain

Elongation of the polypeptide chain begins by the appropriate aminoacyl-tRNA binding to the codon in the A site of the ribosome.

Termination of Translation

At a stop codon, a release factor reads the triplet, and polypeptide synthesis ends; the polypeptide is released from the tRNA, the tRNA is released from the ribosome, and the two ribosomal subunits separate from the mRNA.

Polysomes

Several ribosomes can translate a mRNA at the same time, forming a polysome.

More than one ribosome can translate a mRNA at one time, making it possible to produce many polypeptides simultaneously from a single mRNA

Mismatch repair

When an incorrect nucleotide is added to the growing strand, replication is STALLED by the fact that the nucleotide's exposed 3′-OH group is in the "wrong" position.

proof-reading

Gamma and delta polymerase proofread the whole of the genome

Gamma is the only polymerase in mitochondria - has to do both functions and give a daughter cell that has high fidelity to the parent

fixes about 99% of these types of errors, but that's still not good enough for normal cell functioning.

strand-slippage

Can get permanent changes in DNA due to wobble-induced/replication error leading to strand-slippage

where

As part of gene, there is the 5' UTR which will be transcribed but not translated as the first ATG needs to be found

Introns are excised during RNA maturations

Spaces of bases between 1 gene and the next. Intergenic means its not coding for a gene

If you get mutations within red arrows, pottentail problems may occur

If theyre upsteam (green ones), in the intergeneic regions, there may not be a problem in that protein

However if its within the promoter region or within very specific sites between the zone and the intron borders, then it could have a very big effect on the expression of that particular gene

Pol II promoter elementa

This is important because if there is a mutation within the promoter, it will be dependent on where in the promoter it has happened

If its within the intergenic regions between the tata and CAAT box, the chances of it having a big effect is minimal

However, if its in one of those elements, it has a greater chance of having an impact on whether that gene will be transcribed

If the mutation is at the transcriptional start size, there is a 100% chance that it will affect the gene expression of that gene

induced physical damage to DNA

Environmental agent(s)

Exposure to chemicals UV rays

spontaneous physical damage to DNA

without any exposure to any environmental agent

Spontaneous biochemical reactions taking place within the cell

Wobble-induced/replication errors

point mutations

single base pair substitutions

1. transition

2. transversion

transition substitions

Purine REPLACED BY A Purine

Adenine = Guanine

Pyrimidine REPLACED BY A Pyrimidine

Thymine = Cytosine

Transversion substitions

purine being replaced by a pyrimidine pyrimidine being replaced by a purine

A/T = G/C

silent mutations

no change in the protein sequence

missense mutations

change the amino acid sequence

protein is okay

nonsense mutation

Introduces a stop codon to early

frameshift missense mutation

A base is excised from the parent strand = produced RNA is one base shorter than parent

Everything has shifted so codons have changed = frame shift

Original stop codon has been removed so it has to carry on until another is found = different protein to the one that was suppose to be made

packaging

DNA is packaged into chromatin

The DNA fundamental unit of chromatin is the nucleosome

Nucleosome, the fundamental unit of chromatin, consists of histones

histones

The MOST common NUCLEAR proteins.
Account for almost half of the proteins isolated from nuclei.

Histones broken down into DNA, which is what is being packaged, proteins, which are going to form part of the nucleosome and a small amount of non-coding RNA which helps keep that transcriptional regulation of genes in play

1:1 ratio of amount of histones to DNA mass that’s present

Non-histone components needed to allow high-level packing of DNA

histone family of proteins

Histones are small and are highly positively charged. Also highly conserved

Very highly conserved histones have a very significant role within cell

H3 & H4 are two of the most highly conserved proteins that a eukaryotic cell has

Followed closely by H2A and H2B, which are slightly more diverse

 but still highly conserved compared to other crucial enzymes in nucleus

H1 shows more divergence but yet still conserved

DNA double helix

NA does not exist as helix in nucleus

Only when acts as a linker between adjacent nuclear sites

DNA is packaged at all times

From 10nm fibre to 30nm fibre up to 300 through to 700

Highly compacted chromatin when we hit metaphase and then the splitting of chromosomes so that we can transfer the genetic information from one cell generation to the next

So by being highly packaged, the genome is protected during the mitotic phase

basic unit of chromatin is the nucleosome

FOUR CORE Histones: H2A, H2B, H3, H4 - Make up nucleosome, 2 molecules of each which make up central optometric core

ONE LINKER Histone: H1 - 1 molecule that is associated with octameric core

146bp of DNA LEFT HANDED SUPERHELIX. DNA path = 1.8 superhelical turns Wrap around nucleosome

Level 1 of DNA Packaging: The Nucleosome

146 base pairs wrap around octamer, histone's central core

And then DNA needs to link from one nucleosome to the next

= around 200 base pairs

Mass of DNA : mass of protein

Heterodimerization of histones H3 and H4

Structure causes histone fold. They fold together and clamp onto each other to make 'handshake' motif, which is very specific to the nucleosome

2 molecules of H3 and H4 which associate together = 2 pairs of 'handshakes' - join together in centre of optometric core to make horseshoe shape in the centre of the nucleus

Histones H2A and H2B form a Dimer

A pair of H2A and H2B binding together in 'handshake' but this time the nucleosome of H3 and H4 are in the centre so H2A and H2B bind above and below

This gives an optometric core around which DNA is wrapped in 1.8 super helical turns and packaging 146 base pairs

If a histone should ‘be altered’ within the octamer then a change in the path of the DNA around the octamer occurs and this may result in a change in the ‘packaging’ of the DNA.

histone variants examples

In females, one of the X chromosomes is inactivated randomly but it must remain inactive and for that to happen, it is made different frim the active X chromosome by pushing away the normal H2A and filling throigh those nucleosomes with macroH2A

When DNA is damaged, there is a break because the DNA is packaged in new Which is signified by the expulsion of the normal H2A and replaced bt H2A.X

H2A v H2A.Z

H2A.Z has only a 60% IDENTITY to H2A

Alters the interaction stability between H2A and H2B

Alters the interaction of the H2A:H2B dimer with H3:H4 tetramer - Interaction is looser which makes the octamer slightly bigger than normal so the DNA wrapped around becomes looser

ALTERs the CANONICAL NUCLEOSOME

H2A.Z containing nucleosomes often associated with transcriptionally active chromatin.

histone variants

Hardly any different in A.A sequence between variants but the subtle differences are sufficient enough to change the normal structure of the tetra in the centre

H3.3 - Important variant when changing the transcriptional process outside of replication

cenpA - Enriched in centromere and telomeres

Variants of histones H3 and H2A differentiate chromatin at CENTROMERES, ACTIVE GENES and HETEROCHROMATIN

Replacement of H3 with H3.3 MARKS actively transcribed loci by replication independent nucleosome assembly

Epigenetically silenced chromatin is enriched or depleted in an abundance of diverse H2A variants

level one packaging

the nucleosome

Four CHs + H1 packages 146bp + linker DNA = ~200bp DNA

DNA makes two turns around the core Forming a LEFT-handed super helix.

DNA path = 1.8 superhelical turns.

DNA does NOT follow a smooth Path around the nulceosome.

Accessibility of Nucleases restricted

level two - 10nm fibre

Lowest level of packing in nucleus

This is when DNA is packaged and each nucleosome is holding onto the next

Double helix is present in linker DNA but is connected to another nucleosome

PACKING RATIO: 6-7

level three - 30nm solenoid

Histone H1 is ESSENTIAL for these higher order forms of packaging

10nm is coiled to form 30nm

Requires 6 nucleosome to coil

PACKING RATIO: ~40

level four - 300nm solenoid

Each loop contains 60-100 kb of DNA tethered by nonhistone scaffold proteins

Bind each loop to scaffold

Loops are well contained and tethered at each end of the loops onto the scaffold so it can be accessed later on

The 30nm solenoid further coils probably employing a PROTEIN SCAFFOLD to condense the DNA still further.

PACKING RATIO: 680

level five - 700nm fibre

300nm coiled again - each loop is a 300nm fibre

Results in a tightly compacted DNA associated each time with nucleosomes going through different packaging levels

The next step is described as the COILED COIL and requires the loops of chromatin to coil again to form a condensed 700nm fibre.

PACKING RATIO: 10^4

level five - 700nm solenoid Chromosome

PACKING RATIO: 10^4

level 6: Metaphase Chromosome

Highest form of packaging

Visible under light microscope

Heterochromatin

Constitutive or Facultative

Highly condensed and in general transcriptionally INACTIVE

Contains no gene and present in telomeres and at the centromere

In most cells 90% of the genome is Inactive BUT only 10% is in this highly condensed form.

Restricted regions of chromosome

Repetitive DNA Sequences

Replicated LATE in S phase

constitutive heterochromatin

All cells of a given species will package the same regions of DNA into constitutive heterochromatin

Genes contained within the constitutive heterochromatin will be poorly expressed due to high packaging

remains CONDENSED most of time in all cells (e.g. Y chromosomes)

euchromatin

contains genes

Lightly stained regions of chromosomes

More ‘open’ chromatin configuration during interphase.

Replicated early in S phase

Contains both transcriptionally active and inactive genes

Differential histone modifications

ACTIVE v INACTIVE
Contains both transcriptionally active and inactive genes

faccultative heterochromatin

DNA packaged in facultative heterochromatin will NOT BE CONSISTENT within the cell types of a species

is regulated and is often associated with morphogenesis or differentiation.

gene is randomly inactivated between the cells. (e.g. X inactivation)

X-Chromosome Inactivation

A sequence in one cell that is packaged in facultative heterochromatin (genes poorly expressed) MAY be packaged in EUCHROMATIN in another cell (and the genes expressed).