TOPICS IN (NANO) BIOTECHNOLOGY

Lecture 3

16th October, 2006

PhD Course

Let’s recap...

Let’s recap...

Let’s recap...

Let’s recap...

Let’s recap...

Replication is the reproduction of genetic material and is semi-conservative

Replication

DNA strands separate at the replication fork

Replication

Replication

Replication_Movie_1

Replication_Movie_2

Replication_Movie_3

Replication_Movie_4

Replication_Movie_5

Telomeres• What about synthesis at the ends of Chromosomes?

• Synthesis can proceed to the end on the leading strand but NOT the lagging strand

• There is no 3’-OH group to add

• So, each successive round of replication would shorten the chromosome by the length of the RNA Primer

• An enzyme, telomerase, has helped us solve this problem

Telomeres and aging• The main function of telomeres is to prevent genomic instability

• The enzyme telomerase, as we saw, helps maintain telomere length

• Telomerase is not active in somatic cells, but highly active in germline and tumor cells

• Mechanisms of aging, or cell senescence, are poorly understood but are a hot topic of research

• Primary mechanism appears to be an increased expression in stress protection genes rather than a lowered metabolism

Telomeres and aging• 4 decades ago, Hayflick and Morehead determined that cells in culture have a limited number of cell divisions they can undergo called the Hayflick Limit

• The purpose of senescence is still unclear

• Studies involving mutations that result in increased life span

Telomerase• Bodnar et al., 1998 showed that activation of telomerase in human cells in culture, led to a substantial increase in their life spans

• Conclusion: Telomere shortening plays a major role in cell senescence.

• Thus, telomere’s appear to be a “clock” that counts the number of times a cell divides

• Telomere shortening leads to activation of other “stress” response genes such as p53 and p21 which stop growth

• Telomerase inhibitors as a therapy for cancer

Telomere repeats• In mammals, chromosome telomeres consist of many tandem repeats of sequence 5’ –TTAGGG – 3’

• Telomeres cap the chromosome ends protecting the gene containing portion of chromosomes

• Telomeres shorten with each mitotic cell division

• Loss of the telomeric repeats leads to chromosome instability

Gene expression• Gene expression occurs by a two-stage process:

• Transcription:

• Single strand of DNA is converted to messenger RNA

• Translation:

• The nucleotide sequence of mRNA is converted into the sequence of amino acids comprising a protein

Gene expression

Possible amplification at each of 2 steps allows for different levels of different proteins

RNA in gene expression

Transcription

• Transcription describes synthesis of RNA on a DNA template

• Messenger RNA is synthesised by the same process of complementary base pairing used to replicate DNA

• The messenger RNA is complementary with the sequence of one DNA strand and identical to the other DNA strand of the duplex

Transcription

Transcription

Transcription_Movie

Eukaryotic_Transcription_Movie

Transcription

RNA Processing• A very important part of translation is RNA splicing.

• This has an important function of getting rid of introns – ‘junk’ DNA…but the role of introns?

• Postulated to be flexible and can control ‘shuffling’ of exons to allow for evolutionary change.

• Post genome research has also shown sequences in the intron areas to be important for disease diagnostics e.g. Long QT syndrome

RNA Processing

RNA Splicing

RNA Splicing

Three important sequences for intron removal

RNA Splicing

Intron forms a lariat

RNA Splicing

RNA splicing performed largely by RNA molecules instead of proteins

These RNA molecules = small nuclear RNAs (snRNAs) that recognize intron-exon boundaries

These interact with proteins to form small nuclear ribonucleoprotein particles = snRNPs

These snRNPs (“snurps”) form the core of the spliceosome, which performs RNA splicing in the cell

RNA SplicingOne role of the snRNAs in the snRNPs is to pair (via complementary base-pairing) with the three special sequences of the introns

The snRNPs then bring together the two ends of the intron

The spliceosome also includes other proteins 50 nm

RNA Splicing

RNA Splicing

Positive consequences of the intron-exon arrangement:

1) Presence of introns in between protein-coding regions makes genetic recombination between exons of different genes more likely

Many present-day proteins represent a patchworks composed from a common set of functional domains, hooked together in differing arrangements

Allowed new proteins to evolve more quickly

RNA Splicing

Positive consequences of the intron-exon arrangement:

2) Allows present-day eukaryotes to pack more information into every gene via alternative splicing of transcripts

Thus, different proteins can be made from the same gene ---depending on cell type ---depending on stage of development

RNA Splicing

RNA Splicing

Mature eukaryotic mRNAs are selectively exported from the nucleus

Cell needs to distinguish between completely processed mRNAs and overwhelming amount of debris generated by RNA processing---only a small fraction of RNA in the nucleus is useful to the cell

Answer: export to the nucleus is highly selective, and is coupled to correct processing

RNA Splicing

The nuclear pore complex recognizes and transports only completed mRNAs

To be “export ready”, the mRNA must be bound to appropriate set of proteins:

1) poly-A-binding protein

2) cap-binding complex

3) proteins that mark completed RNA splices

It’s the entire set that marks the mRNA as ready

RNA Splicing

A specialized set of RNA binding proteins mark a mature mRNA as “export ready”

RNA_splicing_movie