You are on page 1of 51

What’s So Special About DNA?

 

DNA is one of the most boring macromolecules imaginable; it is made of only four building blocks and has a perfectly monotonous structure. Worse yet, DNA just sits there - it doesn’t catalyze reactions or build the cell or organism. So, what’s so good about DNA? The answer lies in DNA’s ability to store and copy information.

How Can DNA Store & Copy Information?

Key properties that allow these neat tricks are that DNA is a double stranded molecule held together by complementary bases that pair through simple rules. DNA is also capable of occasional change, and occasionally, change is good.

What’s a Gene?

Such a simple question, such a complicated answer. In part, the answer depends on what level (molecular, cellular, organismal) we’re interested in. A working definition (at the molecular level) - A gene is a sequence of DNA capable of producing some element of biological function. Biological function can refer to many things. It may, for example be a readily observable trait, like skin color, a cellular property, the length of the cell cycle, or a molecular biological property, like the

Eukaryotes

Mammalian genome contains at least 35000 individual genes, which together account for less than 20% of the DNA Function of the remaining DNA ( junk ) sequences is not yet clear

Anatomy of a Gene

Prokaryotic  circular chromosomes  polycistronic- one gene can code for more than one protein  genes can be transcribed together Eukaryotic  on many chromosomes  monocistronic  made of exons & introns

Variability in point mutation inβ globin gene
Promote r defects
EXON 1
CACCC ATAA CCAAT

Nonsense & frameshift mutations
EXON 2 EXON 3

IVS I

IVS II Poly A consensus mutation

RNA processing defects

DNA strands
Each gene ( segment of DNA ) is made of two complementary strands ( A=T, G≡C )  One strand ( sense strand ) contains the specific base sequence which code for protein sequence  The other ( template ) is complementary to the sense strand & us used to synthesize the mRNA

Anatomy of a Gene

Promoters

Promoters are characteristic sequences of DNA, usually located in front ( upstream) of the gene that is to be transcribed. In prokaryotes , simplest promoter found in which two general sequences are found:
 

One (6-8bp) located at -35bp upstream & function in initial binding of RNA polymerase Second sequence (6-8 ) rich in A-T bases and located at -10bp upstream ( TATA box 0r pribnow box) helps in dissociation of DNA strands

In eukaryotes promoters are more complicated, in addition there are additional sequences which are known as

Middle Repetitive DNA
))1 Some middle repetitive DNA consists of genes that specify transfer of RNAs, or histone proteins that are required in large amounts in the cell. Other middle repetitive DNA sequences have no known useful function, but as they represent a significant portion of the genome, they may participate in chromosomal rearrangements.

Middle Repetitive DNA
))2
The best characterized repetitive sequence in humans is known as the Alu sequence. Between 300,000 and 500,000 Alu 1 repeats of about 300 base pairs each exist in the human genome, comprising 3-6% of the total genome. Individual repeats of the Alu sequence may vary by 10-20% in identity. Alu sequences also occur in monkeys and rodents, and similar sequences occur in slime molds and other animal phyla. In addition to the Alu family, there are several other families of middle repetitive DNA in the

RNA
  

Contains the bases G, A, C & U Single stranded Many types 1. mRNA which is direct carrier of genetic information (constitutes 3% of total cellular RNA) 2. rRNA, many types,5S,18S,5.8S (constitutes 80% of total cellular RNA) 3. tRNA, carrier of activated amino acids & has an anticodon for binding to mRNA 4. Other types include hnRNA, snRNA

RNA polymerases (Eukaryotic)
   

RNA poly 1- synthesis of rRNA RNA poly 11- synthesis of mRNA RNA poly 111- synthesis of rRNA & tRNA All have the following characteristics:  Synthesize in 5’ – 3’ direction  Template dependent & no requirement for primer  Large multisubunit enzymes  Initiates polymerization at the promoter region  Hollo enzyme = core enzyme + sigma factor

Base pairing in transcription

Basic & Not so Basic Transcription

The coding strand (sense) carries the code, while the template strand (antisense) is the strand read by RNA polymerase to make the 5’-3’ transcript. Hollo enzyme is the entire enzyme (5 subunits) including sigma factor. Sigma factor enables the polymerase to recognize the promoter regions & will be released following polymerase

Transcription

nucleus

Transcription

nucleus

cytosol

Transcription

Requires the following
1.

2.

3.

DNA directed polymerases Transcription ( sigma factor, TF11A ….) dNTP

RNA factors TF11D,

Mechanism of Transcription

Initiation
 

Recognition of promoter region Binding at the promoter region Bond formation Translocation Rho independent termination Rho dependent termination

Elongation
 

Termination
 

Transcription

Initiation

Involves the interaction of the RNA polymerase with DNA at a sitespecific (promoters ) fashion,so that a correct sequence of DNA can be used as a template. Initiation in eukaryotes is far more complicated.

 Transcription
Binding   RNA polymerase binds to promoter  = specific DNA sequence that determines where RNA polymerase binds and starts transcription 

 Transcription
Binding 

RNA polymerase binds to promoter  = upstream (5 ') of transcription unit Determines which DNA strand is template 

Elongation

Once RNA polymerase has bound to a promoter, it begins selecting appropriate complementary ribonucleotide and forming phosphodiester bridges between the nucleotide and the nascent chain Elongation is very rapid, occurring at the rate of 40nt per second. The double stranded DNA must be continually unwound, so that the template strand is accessible

Termination

In prokaryotes, occurs by one of two mechanisms:
Rho-independent: A hairpin loop is formed just before a sequence of six to eight uridine (u) residues near the 3’ end of the newly synthesized RNA. This secondary structure dislodges the RNA polymerase from DNA template, resulting in termination. Rho-dependent: Requires the action of a protein factor called Rho, which has an ATPdependent helicase activity. The Rho protein is believed to travel along the newly synthesized RNA, chasing the RNA polymerase and dislodges the RNA

Post-Transcriptional Modification

In Eukaryotes large primary transcripts must be processed to a smaller size before leaving the nucleus. In prokaryotes processing may involve either removal of sequences from primary transcript or removal and rejoining of segments of the transcript. This process requires endoribonucleases and exoribonucleases. In Eukaryotes, spliceosomes are required.

RNA Processing

mRNA Processing 

Capping

atypical nucleotide added to the 5’ end of the transcript
7’ methylguanosine  linked by 5’-5’ triphosphate bridge to the 5’ end of the RNA

mRNA Processing -

Splicing

mRNA Processing Tailing  PolyA Polymerase Polyadenylation

PolyA Polymerase Polyadenylation )eukaryotes) Eukaryotic transcripts have polyA tails. These arise due to cleavage and addition of AAAAs. The signal for cleavage and polyadenylation is AAUAAA. Factors involved: Cleavage and Polyadenylation Specificity Factor CPSF Cleavage Stimulatory Factor CStF

mRNA Processing -

Tailing

Similar   Complementary template  TEMPLATE STRAND 

Replication vs Transcription

 

Similar   Complementary template   Synthesis 5 ' to 3 ' (add only to 3 ' end)   

Replication vs Transcription

Similar   Complementary template used   Synthesis 5 ' to 3 ' (add only to 3 ' end)   Triphosphate precursors provide energy for  polymerization 

Replication vs Transcription

Different   Deoxyribonucleotide precursors vs ribonucleotide   

Replication vs Transcription

Different  Deoxyribonucleotide precursors vs ribonucleotide   DNA polymerases proofread 

Replication vs Transcription

Different   Deoxyribonucleotide precursors vs ribonucleotide   DNA polymerases proofread   DNA synthesis is discontinuous on the lagging  strand 

Replication vs Transcription

Different  All DNA is duplicated once/ cell cycle during  DNA synthesis 

Replication vs Transcription

 

Ribozymes RNAs that act like enzymes (1)
In some instances, RNAs have a catalytic ability similar to the type of activities previously ascribed only to proteins. These special molecules, known as ribozymes, possess a catalytic activity and a substrate specificity similar to those of proteinaceous enzymes. The substrate specificity of a ribozyme is determined via nucleotide base pairing between complementary sequences contained within the enzyme

Ribozymes RNAs that act like enzymes (2)
Just like enzymes that are proteins, the ribozyme will cleave its substrate RNA at a specific site and then release it, without itself being consumed in the reaction.Ribozymes are being considered as possible therapeutic agents for diseases that are caused by the inappropriate expression of an RNA or the expression of a mutated RNA. In these cases, the development of a ribozyme that had specificty for a particular RNA could result in the seletive degradation of the substrate, eliminating it from the cell

End Of Show