dna replication (semi-conservative method) molecular biology – dna replication, transcription

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DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY DNA replication, transcription

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Page 1: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

DNA REPLICATION(semi-conservative method)

MOLECULAR BIOLOGY – DNA replication, transcription

Page 2: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-2 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

REPLICATION FORK

1000 nt / sec !

Meselson-Stahl experiment

The enzyme DNA polymerase uses the two parental strands as a template to faithfully synthesise new daughter strands according to the specific Watson & Crick

base-pairing system (A-T and G-C)

Page 3: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-14 Molecular Biology of the Cell (© Garland Science 2008)

Unwinding and strand separation achieved by DNA helicase using

the enegy released from ATP hydrolysis

MOLECULAR BIOLOGY – DNA replication, transcription

Semi-conservative replication by DNA polymerase requires that the

two anti-parallel parental DNA strands are unwound to give a

single stranded templates

Page 4: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-16 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Secondary structure in the single stranded template can hinder DNApol

Single-strand binding proteins facilitate

DNApol’s progress

Page 5: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-3 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Incoming deoxyribonucleotide trisphosphates or dNTPs(i.e. dATP, dCTP, dTTP or dGTP depending on base pairing with

TEMPLATE STRAND)

Newly synthesised strand replicated in a 5’ - 3’

direction

New dNTPs are added to the free 3’OH group of preceding nucleotides in the

DNA strand by means of a condensation reaction thus forming a

new phosphodiester bond

Pyrophosphate and water are by-products

Page 6: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

UDP deoxyUDP dTTPADP deoxyADP dATPGDP deoxyGDP dGTPCDP deoxyCDP dCTP

dTDP

ribonucleotidereductase

kinase

Methotrexateanti-cancer drug

MOLECULAR BIOLOGY – DNA replication, transcription

Folate cofactor only obtainable as a dietary supplement(extra supplements for children and pregnant women)

Synthesis of dNTP substrates required for DNA replication

Page 7: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-18c Molecular Biology of the Cell (© Garland Science 2008)

DNApol III

MOLECULAR BIOLOGY – DNA replication, transcription

Chromosomal DNA synthesis catalysed by DNA polymerase

III (DNApol III)

DNApol III requires the help of ‘sliding clamp’ in order to bind

DNA and start replication

The sliding clamp however requires a complex of proteins (i.e. the

‘clamp loading complex’) plus the energy released from ATP hydrolysis

to be loaded onto the DNA

DNA synthesis can now occur in 5’ - 3’ direction although any base-

pairing mistakes can be corrected by removing the incorrect base via the ‘3’-exonuclease activity’ of the

DNApol III complex

Page 8: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

replication direction

3’

3’

5’

5’

3’leading strand

3’5’

5’ 5’3’

lagging strand

DNA polymerase synthesizes in 5’ 3’ direction therefore the two newly synthesized daughter strands are made

differently

MOLECULAR BIOLOGY – DNA replication, transcription

Okazaki fragmentsare eventually joined (ligated) together to form a complete strand

Page 9: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-11 Molecular Biology of the Cell (© Garland Science 2008)

DNA polymerase III can not simplystart synthesizing a new strand

It can only elongate from existing one (i.e. a free 3’OH group is

required)

MOLECULAR BIOLOGY – DNA replication, transcription

How does DNA synthesis get started?

A specialized RNA polymerase called ‘DNA primase’ can simply start the synthesis of a new strand using the

template DNA strand as a guide

These 11-12 nucleotide RNA primers then provide the free 3’OH required by DNApol III to replicate the rest of

the DNA

Page 10: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

MOLECULAR BIOLOGY – DNA replication, transcription

DNApol I

What happens to the RNA primers and Okazaki fragments

on the lagging strand?

The ‘gap’ between the Okazaki DNA fragment and the RNA primer is

recognised by ‘DNA polymerase I’ that then removes the RNA primer and fills in the space with template

directed DNA

Lastly the two adjacent DNA Okazaki fragments are joined by the enzyme

‘DNA ligase’

DNApol III completes the synthesis of the DNA Okazaki fragment up until

the previous RNA primer without joining the two molecules together

Figure 5-12 Molecular Biology of the Cell (© Garland Science 2008)

Page 11: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-19a Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

DNA Replication Summary

N.B. the lagging strand template is bent round so both DNApol’s proceed in the same direction!

Page 12: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-19b,c Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Detailed electronmicrograph of a bacterial DNA replication fork

Page 13: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-6 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Electronmicrograph of two replication forks progressing around a circular bacterial DNA genome

Page 14: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-25 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Summary of bidirectional

replication forks with leading and lagging strand

synthesis

Page 15: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-21 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

The action of helicase at the replication fork places great strain on the DNA double helix ahead of

it because the two ends of the helix cannot freely rotate with

respect to each other

Page 16: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-22 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

An enzyme called ‘DNA topisomerase I’ relieves this strain by catalysing a break in the phosphodiester backbone of one DNA strand

allowing the two ends of the helix to rotate relative to each other

Page 17: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

MOLECULAR BIOLOGY – DNA replication, transcription

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter11/animation_quiz_2.html

DNA replication summary

Page 18: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-26 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Relatively small bacterial genomes

are circular and are usually

replicated from a single ‘replication

origin’ that consists of

tandem repeat rich DNA

sequences N.B. bi-directional replication from a single replication

origin.

Known as OriC in E-coli.

Page 19: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-34 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Large eukaryotic genomes (multiple linear chromosomes) are replicated from many different ‘origins of replication’

• Multiple origins of replication comprising many different sequence variations (approx 100 000 in human genome)

• Not all activated at the same time

• Mechanism involves the assembly of the ‘pre-replication complex (pre-RC)’ of proteins prior to their activation and initiation of DNA synthesis

Page 20: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

During DNA replication there is aProblem with the end of the lagging strand:

... progressive shortening of chromosomal ends and genetic instability

MOLECULAR BIOLOGY – DNA replication, transcription

Page 21: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

TTAGGGTTAGGGTTAGGGTTAGGG

(TTAGGG)20-HUNDREDS

Telomeres made of many repeatsSpecies Repeat Sequence

Arabidopsis TTTAGGG

Human TTAGGG

Oxytricha TTTTGGGG

Slime Mold TAGGG

Tetrahymena TTGGGG

Trypanosome TAGGG

Yeast (TG)1-3TG2-3

MOLECULAR BIOLOGY – DNA replication, transcription

Chromosome ends comprised of TELOMERES

Telomeres provide a kind of ‘buffer’ for the chromosomal ends that protect genes located in the ‘sub telomeric’ regions

Therefore only telomeric sequence is lost during DNA replication

HOW ARE TELOMERES MAINTAINED?

Page 22: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 5-41 Molecular Biology of the Cell (© Garland Science 2008)

TELOMERES ARE ELONGATED BY ACTION OF TELOMERASE

MOLECULAR BIOLOGY – DNA replication, transcription

Conventional DNA synthesis is achieved by RNA priming of the elongated parental strand thus maintaining

telomere length and chromosome integrity

Page 23: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

• ~ 150 genes control telomere length in yeast and shortening telomeres is associated with cell senescence and death

• telomerase highly active in >90% of tumors i.e. cell immortalization and uncontrolled proliferation (drug target)

• many adult cell types have detectable telomerase activity, but it is a highly regulated, fine tuned activity

MOLECULAR BIOLOGY – DNA replication, transcription

Page 24: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Correlation between levels of perceived stress and telomere length and action of telomerase

MOLECULAR BIOLOGY – DNA replication, transcription

RELAX AND KEEP YOUR TELOMERES LONG!

Page 25: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

MOLECULAR BIOLOGY – DNA replication, transcription

Telomere and Telomerase video/ tutorial

http://www.youtube.com/watch?v=AJNoTmWsE0s

Page 26: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

AUG UAA

double stranded DNA

TRANSLATIONprotein coding sequence

or open reading frame

Functional Protein

MAPSSRGG…..

MOLECULAR BIOLOGY – DNA structure, genetic code

TRANSCRIPTION

ATG GCT CCT TCT TCC AGA GGT GGC . . . . . . TAATAC CGA GGA AGA AGG TCT CCA CCG . . . . . . ATT

5’5’3’

3’

single stranded mRNA AUG GCU CCU UCU UCC AGA GGU GGC . . . . . . UAA

TRANSCRIPTION

Focus on how the genetic information contained within DNA is copied into

RNA and it’s consequences

Page 27: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 6-21 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Eukaryotic mRNAs are not synthesised in a form that can be immediately

translated to protein

i.e. there are processing and translocation steps

RNA processing

translocation

Page 28: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 6-22a Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Polycistronic mRNA(e.g related gene operons)

Monocistronic mRNA

Page 29: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Table 6-1 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Page 30: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

TRANSCRIPTION INITIATION

MOLECULAR BIOLOGY – DNA replication, transcription

How does the RNA polymerase recognise the the correct place within DNA to start transcribing mRNA for a gene?

Doubel stranded DNA

RNA polymerse complexSingle stranded mRNA

Page 31: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 6-11 (part 1 of 7) Molecular Biology of the Cell (© Garland Science 2008)

TRANSCRIPTION START IN PROKARYOTES

MOLECULAR BIOLOGY – DNA replication, transcription

Molecular interaction with the ‘sigma () factor’ permits RNA polymerase (designated ‘holoenzyme’) to bind DNA at specific regions called

‘promoters’

The core ‘RNA polymerase complex’ (consisting of & ’catalytic and 2x regulatory subunits) alone is unable to recognise and bind

DNA

Page 32: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 6-12a Molecular Biology of the Cell (© Garland Science 2008)

GENEPROMOTER

MOLECULAR BIOLOGY – DNA replication, transcription

+1

1st ribonucleotide to be incorporated into

mRNA

The sigma factor recognises ‘consensus’ sequences in the promoter DNA

There are x2 consensus sequences at the -35 and -10

positions of the DNA

-35 -10

Variations in the -35 & -10 DNA sequences of different gene promoters affect how often mRNA synthesis can be initiated. Additionally bacteria have multiple sigma factors each with subtle

differences in binding affinity for -35 & -10 sequence variants.

The sigma factor recognises -35 & -10 and positions the

RNA polymerase in the correct position to start

synthesis of mRNA starting from the +1 position

-35

-10 +1

Page 33: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

MOLECULAR BIOLOGY – DNA replication, transcription

After initiation, sigma factor disassociates and synthesis of the new mRNA molecule occurs in a 5’ - 3’ direction i.e.

‘elongation phase’

The mRNA sequence is specified by base-pair complementarities with the ‘template/ non-coding/ antisense’ DNA strand and therefore is a copy/

‘transcript’ of the ‘non-template/ coding/ sense’ strand wit U replacing T

5’

3’

5’

Page 34: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

The repeats are copied into the transcribed mRNA and form a ‘hairpin

loop’ that is sensed by the RNA polymerase causing it to pause/ stutter.

Weak hydrogen bonding between the run of A nucleotides in the DNA template strand and the U ribonucleotides of the mRNA cause the disassociation of the whole RNA polymerase complex i.e.

‘intrinsic termination’

In other cases the binding of special helicases called ‘Rho ()proteins’

facilitate the disassociation i.e. ‘rho-dependent termination’

Just as promoter sequences in DNA instruct where transcription should begin, other DNA sequences called ‘terminators’ specify where it should stop.

MOLECULAR BIOLOGY – DNA replication, transcription

How does RNA polymerase know when to stop?

‘terminators’ sequences are inverted DNA repeats followed

by a run of A nucleotides

Page 35: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

MOLECULAR BIOLOGY – DNA replication, transcription

Prokaryotic transcription cycle video/ tutorial

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter12/animation_quiz_1.html

Page 36: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Table 6-2 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

TRANSCRIPTION IN EUKARYOTES

Additionally transcription occurs in the nucleus and not in the cytoplasm

Page 37: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

i.e. PROTEIN CODING GENE TRANSCRIPTION

Table 6-3 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Recognition of the promoter and the initiation of transcription in eukaryotes is more complex

In addition to RNA polymerase a host of other proteins known as the ‘General Transcription Factors (GTFs)’ are required

GTF’s help RNA polymerase recognise a T/A rich DNA sequence motif in the promoter called the ‘TATA box’, correctly position it on the ‘chromatin template’ with respect to

the +1 position and convert it to form capable of RNA synthesis

i.e. help form a ‘pre-initiation complex’

Page 38: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

MOLECULAR BIOLOGY – DNA replication, transcription

Binding of ‘specific transcription factors’ positively or negatively affect frequency of transcription initiation

• specific transcription factors bind particular DNA sequences only found in the locality of certain genes

• therefore can help regulate which genes mRNAs are synthesised within a cell (e.g. confine the production of antibodies in white blood cells but not in neurones!)

e.g. ‘short-range upstream regulator elements’

e.g. ‘long range enhancer sequences’

Pre-initiation complex of RNA polymerase II and GTFs at TATA box

Specific transcription factor binding

Page 39: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 6-19 Molecular Biology of the Cell (© Garland Science 2008)

CORE PROMOTER

MOLECULAR BIOLOGY – DNA replication, transcription

Page 40: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

MOLECULAR BIOLOGY – DNA replication, transcription

Initiation of eukaryotic transcription video/ tutorial

http://bcs.whfreeman.com/thelifewire/content/chp14/1402002.html

Page 41: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

RNA PROCESSING in EukaryotesMOLECULAR BIOLOGY – DNA replication, transcription

Eukaryotic genes typically consist of segments of protein coding DNA sequence called ‘exons’ that are interspersed with non-protein coding sequences called ‘introns’ (that are often very long).

Unlike prokaryotes, the DNA sequence encoding eukaryotic protein is not often organised in one continuous length that

when transcribed is a fully functional mRNA that can be translated into protein. The mRNA first needs to be

‘processed’

• both the exons and introns are transcribed by RNA polymerase II to yield long RNA molecules called ‘Pre-mRNAs’

Pre-mRNA• Pre-mRNAs are modified (co-transcriptionally) by addition of a 5’-cap and 3’ polyadenylation motifs (important for stability and translation).

CAP AAA(n)

• the sequence corresponding to the introns is removed from the pre-mRNA in a process called ‘SPLICING’ to yield a ‘mature mRNA’

SPLICING

Mature mRNA CAP AAA(n)

• the ‘spliced’ together protein coding mRNA sequence corresponding to the exons in the DNA can now be translated into function protein (except for ‘untranslated regions/ UTRs’ at the 5’ & 3’ ends

Page 42: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 6-22b Molecular Biology of the Cell (© Garland Science 2008)

Capping Eukaryotic Pre-mRNA

MOLECULAR BIOLOGY – DNA replication, transcription

RNA terminal

phosphatse

‘Enzyme capping complex (CEC)’

Bound to RNApolII complex and caps the pre-mRNA co-

transcriptionally

Leads to the covalent attachment of a ‘7-methylguanosine cap (m7G cap)’ at the 5’ end of the

pre-mRNA via an unusual 5’ - 5’ triphosphate bond

The m7G cap ensures:

• the (pre)mRNA is protected from degradation

• mRNA export from nucleus

• mRNA is translated to give functional protein

Page 43: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 6-38 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Polyadenylation of Eukaryotic Pre-mRNA

Cleavage and polyadenylation specificity factor (CPSF) & cleavage stimulation factor

(CstF)

CPSF & CstF recognise the cleavage and polyadeylation signals once they have been

transcribed into the pre-mRNA

CPSF & CstF binding recuits other factors that cleave the pre-mRNA chain free of the RNA polymerase II complex

Additionally polymerase (PAP) and poly-A-binding proteins

(PABPs) are also recruited to this polyadenylation complex

Polyadenylation importance:

• participates in transcriptional termination

• protects mRNA from degradation

• mRNA export from nucleus

• mRNA translation to give functional protein

PAP extends the 3’ end of the RNA by untemplated addition of up to 200 adenosine (A) nucleotides that are

then bound by PABPs

Page 44: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

MOLECULAR BIOLOGY – DNA replication, transcription

mRNA capping and polyadenylation video/ tutorial

http://www.youtube.com/watch?v=YjWuVrzvZYA

Page 45: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

MOLECULAR BIOLOGY – DNA replication, transcription

SPLICING of Eukaryotic Pre-mRNA

The very precise removal of non protein coding intron derived sequences from pre-mRNAs (N.B. genetic code) is catalysed by the multiple subunit containing complex called the ‘SPLICEOSOME’

Pre-mRNASpliceosome

The subunit composition of the spliceosome changes as the splicing of introns progresses

The main class of subunit are the ‘small nuclear ribonucleoproteins/ snRNPs (“snurps”) ’

snRNPs are complexes of proteins and ‘small nuclear

RNA (snRNA)’

There are 5 different snRNPs designated U1,

U2, U4, U5 & U6

snRNA

Protein

The snRNAs in snRNPs permit spliceosome

assembly

&

Recoginse ‘specific splice signals’ within the pre-

mRNA by complementary base-paring

Page 46: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 6-28 Molecular Biology of the Cell (© Garland Science 2008)

snRNAs of snurps recognize 3 types of splice signal in the pre-mRNA

5’ splice site ‘branch site’ 3’ splice site

MOLECULAR BIOLOGY – DNA replication, transcription

This adenosine is critical to successful

splicing

The recognition of these 3 splice signal sequences

directs the correct assembly of the spliceosome and the correct joining of the exons

Page 47: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 6-26a Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Spliceosome reaction mechanism

Cowboy’s lariat

2’ - 5’ phosphodiester

bond

Page 48: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 6-29 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Where do the snRNPs fit in?

N.B. the dynamics in the composition of the spliceosome between catalysing the first and

second phosphoryl-transfer reactions

Page 49: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 6-30c Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

snRNP & snRNA interactions in the two forms of spliceosome active site

Formation of the lariat with the conserved adenosine (A) in the

branch site (i.e catalysis of the 2’ -5’ phosphodiester bond)

Excision of the lariat and joining of the two exons (i.e. a conventional 3’ -

5’ phosphodiester bond)

Page 50: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

MOLECULAR BIOLOGY – DNA replication, transcription

General splicing video/ tutorial

http://bcs.whfreeman.com/thelifewire/content/chp14/1402001.html

Page 51: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 6-36 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – DNA replication, transcription

Self splicing introns

Some lower eukaryotic species e.g. tetrahymena

Some fungi and plants species

Secondary structure formation within the intron caused by complementary base-pairing between its nucleotides forms inherent enzymatic activities (i.e. ‘ribozymes’) that catalyse the removal of the

intron and the joining of exons

Unicorporated guanine (G)

particpates as a cofactor and intron excised as a linear

moleclue

As with spliceosome assisted splicing a conserved adenine (A) nucleotide in the intron participates in the reaction and the intron is excised as a

lariat

Page 52: DNA REPLICATION (semi-conservative method) MOLECULAR BIOLOGY – DNA replication, transcription

Figure 6-31 Molecular Biology of the Cell (© Garland Science 2008)

ALTERNATIVE SPLICING

MOLECULAR BIOLOGY – DNA replication, transcription

Alternative splicing is a mechanism by which one gene can code for more than one version of a protein depending upon which exons make it into the mature mRNA and are translated.

Therefore provides an evolutionary mechanism for extra diversity!