Central Dogma

Central Dogma

• Central Dogma

• mRNA

• Prokaryote
• Eukaryote
• Extrachromosomal (Mitochondrial)

• tRNA

• Translation

• mRNA Life Cycle

• Polycistronic and monocistronic mRNAs

• Prokaryotic and eukaryotic mRNAs

mRNAs are single stranded RNA molecules

• mRNAs are single stranded RNA molecules

• They are copied from the TEMPLATE strand of the gene, to give the SENSE strand in RNA

• They are transcribed from the 5’ to the 3’ end

• They are translated from the 5’ to the 3’ end

mRNAs are single stranded RNA molecules

• mRNAs are single stranded RNA molecules

• They are copied from the TEMPLATE strand of the gene, to give the SENSE strand in RNA

• They are transcribed from the 5’ to the 3’ end

• They are translated from the 5’ to the 3’ end

mRNAs are single stranded RNA molecules

• mRNAs are single stranded RNA molecules

• They are copied from the TEMPLATE strand of the gene, to give the SENSE strand in RNA

• They are transcribed from the 5’ to the 3’ end

• They are translated from the 5’ to the 3’ end

mRNAs are single stranded RNA molecules

• mRNAs are single stranded RNA molecules

• They are copied from the TEMPLATE strand of the gene, to give the SENSE strand in RNA

• They are transcribed from the 5’ to the 3’ end

• They are translated from the 5’ to the 3’ end

mRNAs are single stranded RNA molecules

• mRNAs are single stranded RNA molecules

• They are copied from the TEMPLATE strand of the gene, to give the SENSE strand in RNA

• They are transcribed from the 5’ to the 3’ end

• They are translated from the 5’ to the 3’ end

mRNAs are single stranded RNA molecules

• mRNAs are single stranded RNA molecules

• They are copied from the TEMPLATE strand of the gene, to give the SENSE strand in RNA

• They are transcribed from the 5’ to the 3’ end

• They are translated from the 5’ to the 3’ end

They can code for one or many proteins (translation of products) in prokaryotes (polycistronic)

• They can code for one or many proteins (translation of products) in prokaryotes (polycistronic)

They encode only one peptide(each) in eukaryotes (monocistronic).

• They encode only one peptide(each) in eukaryotes (monocistronic).

• Polyproteins are observed in eukaryotic viruses, but these are a single translation product, cleaved into separate proteins after translation.

Catalysed by RNA Polymerase

• Catalysed by RNA Polymerase

• Stages: initiation, elongation and termination

• Initiation is at the Promoter sequence

• Regulation of gene expression is at the initiation stage

• Transcription factors binding to the promoter regulate the rate of initiation of RNA Polymerase

mRNA is synthesised by RNA Polymerase

• mRNA is synthesised by RNA Polymerase

• Translated (once or many times)

• Degraded by RNAses

• Steady state level depends on the rates of both synthesis and degradation

• Production  Degradation

mRNA is synthesised by RNA Polymerase

• mRNA is synthesised by RNA Polymerase

• Translated (once or many times)

• Degraded by RNAses

• Steady state level depends on the rates of both synthesis and degradation

• Production  Degradation

mRNA is synthesised by RNA Polymerase

• mRNA is synthesised by RNA Polymerase

• Translated (once or many times)

• Degraded by RNAses

• Steady state level depends on the rates of both synthesis and degradation

• Production  Degradation

mRNA is synthesised by RNA Polymerase

• mRNA is synthesised by RNA Polymerase

• Translated (once or many times)

• Degraded by RNAses

• Steady state level depends on the rates of both synthesis and degradation

• Production  Degradation

Translated from 5’  3’ end in cytoplasm

• Translated from 5’  3’ end in cytoplasm

• Ribosomes bind at 5’ cap, and do require a free 5’ end

• Can contain only one translated open reading frames (ORF).

• Only first open reading frame is translated

Translated from 5’  3’ end in cytoplasm

• Translated from 5’  3’ end in cytoplasm

• Ribosomes bind at 5’ cap, and do require a free 5’ end

• Can contain only one translated open reading frames (ORF).

• Only first open reading frame is translated

Translated from 5’  3’ end in cytoplasm

• Translated from 5’  3’ end in cytoplasm

• Ribosomes bind at 5’ cap, and do require a free 5’ end

• Can contain only one translated open reading frames (ORF).

• Only first open reading frame is translated

Consists of a guanine nucleotide connected to the mRNA via an unusual 5' to 5' triphosphate linkage.

• Consists of a guanine nucleotide connected to the mRNA via an unusual 5' to 5' triphosphate linkage.

Further modifications include the methylation of the 2' hydroxy-groups of the first 3 ribose sugars of the 5' end of the mRNA.

• Further modifications include the methylation of the 2' hydroxy-groups of the first 3 ribose sugars of the 5' end of the mRNA.

• Functionally the 5' cap looks like the 3' end of an RNA molecule (the 5' carbon of the cap ribose is bonded, and the 3' unbonded).
• Offers resistance to 5' exonucleases.

Single stranded

• Single stranded

• Polycistronic (many ORFs)

• Unmodified 5’ and 3’ ends

• Transcribed and translated together

• Show similarity to prokaryote genes

• and transcripts

tRNA first hypothesized by Francis Crick.

• tRNA first hypothesized by Francis Crick.

• Small RNA chain that transfers a specific amino acid to a growing polypeptide chain in the ribosome

• Has a 3' terminal site for amino acids (whose linkage depends on aminoacyl tRNA synthetase).

• Contains a three base region called the anticodon that complements the codon on the mRNA.

• Each type of tRNA molecule can be attached to only one type of amino acid.

• tRNA molecules bearing different anticodons may also carry the same amino acid (degenerecy).

tRNA has primary structure (sequence), secondary structure (cloverleaf), and tertiary structure (L-shape).

• tRNA has primary structure (sequence), secondary structure (cloverleaf), and tertiary structure (L-shape).

• Small RNAs 75 - 85 bases in length

• Highly conserved secondary and tertiary structures

• Each class of tRNA charged with a single amino acid

• Each tRNA has a specific trinucleotide anti-codon for mRNA recognition

• Conservation of structure and function in prokaryotes and eukaryotes

The 5'-terminal phosphate group.

• The 5'-terminal phosphate group.

• The acceptor stem (7bp) composed by the 5'-terminus base paired to the 3'-terminus (contains non-Watson-Crick base pairs).

• The CCA tail is at the 3' end of the tRNA molecule (important for the recognition of tRNA by enzymes critical in translation). NOTE: In prokaryotes, the CCA sequence is transcribed. In eukaryotes, the CCA sequence is added.

• The D arm (18bp) ends in a loop which contains dihydrouridine.

• The anticodon arm (ca17bp) contains the anticodon.

• The T arm (17bp) contains TC sequence ( = pseudouridine).

• Modified (methylated) bases occur in several positions outside the anticodon. First anticodon base sometimes modified to inosine or ).

An anticodon is a unit made up of three nucleotides that complement the three bases of the codon on the mRNA.

• An anticodon is a unit made up of three nucleotides that complement the three bases of the codon on the mRNA.

Different base triplets (codons) in mRNA code for different amino acids.

• Different base triplets (codons) in mRNA code for different amino acids.

The Genetic Code is degenerate

• The Genetic Code is degenerate

The Genetic Code is degenerate

• The Genetic Code is degenerate

• Serine can be: UCU, UCC, UCA, UCG, AGU & AGC

• Each tRNA contains a specific anticodon triplet sequence that can base-pair to one or more codons for an amino acid.

• For example, one codon for Serine is AGU; the anticodon being UCA.

• Some anticodons can pair with more than one codon due to wobble base pairing. When the first nucleotide of the anticodon is either inosine or pseudouridine (can hydrogen bond to different bases).

Aminoacylation is the process of covalently adding an aminoacyl group to a compound tRNA).

• Aminoacylation is the process of covalently adding an aminoacyl group to a compound tRNA).

• Each tRNA is aminoacylated (charged) with a specific amino acid by an aminoacyl tRNA synthetase.

• There is normally a single aminoacyl tRNA synthetase for each amino acid.

• There can be more than one tRNA, and more than one anticodon, for an amino acid.

Organisms have varying amounts of tRNA genes.

• Organisms have varying amounts of tRNA genes.

• C. elegans has 29,647 genes of which 620 code for tRNA.

• Saccharomyces cerevisiae has 275 tRNA genes in its genome.

• In the human genome there are:

• 4,421 non-coding RNA genes (which include tRNA genes).
• 22 mitochondrial tRNA genes
• 497 nuclear genes encoding cytoplasmic tRNA molecules and
• 324 tRNA-derived putative pseudogenes.

Cytoplasmic tRNA genes are grouped into 49 families according to their anti-codon features.

• Cytoplasmic tRNA genes are grouped into 49 families according to their anti-codon features.

• tRNA genes are found on all chromosomes, except 22 and Y.

• High clustering on 6p and 1 is observed (140 tRNA genes).

• tRNA molecules are transcribed (in eukaryotic cells) by RNA polymerase III, unlike messenger RNA which is transcribed by RNA polymerase II.

• pre-tRNAs contain introns; in bacteria these self-splice, whereas in eukaryotes and archaea they are removed by tRNA splicing endonuclease.

Prokaryotic ribosomes are smaller than most of the eukaryotes.

• Prokaryotic ribosomes are smaller than most of the eukaryotes.

• The ribosomes in eukaryote mitochondria resemble those in bacteria.

• The function of ribosomes is the assembly of proteins (translation).

• Ribosomes catalyze the assembly of individual amino acids into polypeptide chains.

• They use mRNA as a template to join a correct sequence of amino acids.

• This reaction uses adapters called tRNA.

First observed in the mid-1950s by Romanian cell biologist George Palade using an electron microscope as dense particles or granules

• First observed in the mid-1950s by Romanian cell biologist George Palade using an electron microscope as dense particles or granules

• Nobel Physiology, 1974.

• The term "ribosome" was proposed by scientist Richard B. Roberts in 1958

• Ribonucleic body (soma)

All ribosomes are composed of two subunits that separate when translation terminates and reunite when an new initiation complex is formed.

• All ribosomes are composed of two subunits that separate when translation terminates and reunite when an new initiation complex is formed.

The basic form of the ribosome is conserved, but there are appreciable variations in the overall size and proportions of RNA and protein in the ribosomes of bacteria, eukaryotic cytoplasm, and organelles.

• The basic form of the ribosome is conserved, but there are appreciable variations in the overall size and proportions of RNA and protein in the ribosomes of bacteria, eukaryotic cytoplasm, and organelles.

Theodor Svedberg (1884-1971).

• Theodor Svedberg (1884-1971).

• Nobel laureate (Chemistry,1926) for his work on colloids and ultracentrifugation.

• Colloid = Mechanical mix (milk) with a dispersed and continuous phase.

• Svedberg, non-SI unit used to characterize the behaviour of a particle in ultracentrifugation.

• S = unit of time amounting to 10-13 s or 100 femtoseconds.

• S not additive, since the sedimentation rate is associated with the shape & size of the particle.

• When two particles bind together there is a loss of surface area, when measured separately they will have Svedberg values that do not add up.

The ribosomal proteins are known as r-proteins.

• The ribosomal proteins are known as r-proteins.

• With the exception of one protein present at four copies per ribosome, there is one copy of each protein.

The ribosomes of higher eukaryotes are larger than those of bacteria.

• The ribosomes of higher eukaryotes are larger than those of bacteria.

• Total content of both RNA & protein is greater

• Major RNA molecules are longer (18S & 28S rRNAs)

• Possess more proteins.

• RNA is the predominant component.

The ribosomes of higher eukaryotic cytoplasm are larger than those of bacteria.

• The ribosomes of higher eukaryotic cytoplasm are larger than those of bacteria.

• Total content of both RNA & protein is greater

• Major RNA molecules are longer (18S & 28S rRNAs)

• Possess more proteins.

• RNA is the predominant component.

The ribosomes of higher eukaryotic cytoplasm are larger than those of bacteria.

• The ribosomes of higher eukaryotic cytoplasm are larger than those of bacteria.

• Total content of both RNA & protein is greater

• Major RNA molecules are longer (18S & 28S rRNAs)

• Possess more proteins.

• RNA is the predominant component.

In prokaryotes:

• In prokaryotes:

• Ribosomes = 2.5 MDa
• rRNA = 66% of the ribosomal mass

• In eukaryotes (mammals):

• Ribosomes = 4.2 MDa
• rRNA = 60% of the ribosomal mass

• Organelle ribosomes are distinct from eukaryotic cytosol ribosomes and take varied forms.

Prokaryotic Ribosome is 70S and composed of Large (50S) and Small (30S) subunits.

• Prokaryotic Ribosome is 70S and composed of Large (50S) and Small (30S) subunits.

Eukaryotic Ribosome is 80S and composed of Large (60S) and Small (40S) subunits.

• Eukaryotic Ribosome is 80S and composed of Large (60S) and Small (40S) subunits.

The ribosomes of chloroplasts & mitochondria also consist of large and small subunits bound together with proteins into one 70S particle (as prokaryotes do).

• The ribosomes of chloroplasts & mitochondria also consist of large and small subunits bound together with proteins into one 70S particle (as prokaryotes do).

Ribosomes are about 20nm in diameter.

• Ribosomes are about 20nm in diameter.

• Can be studied with EM.

The complete 70S ribosome has an asymmetric construction.

• The complete 70S ribosome has an asymmetric construction.

• The partition between the head and body of the small subunit is aligned with the notch of the large subunit, so that the platform of the small subunit fits into the large subunit.

• There is a cavity between the subunits which contains important sites.

Classified as "free" or "membrane-bound".

• Classified as "free" or "membrane-bound".

• Free ribosomes move about the cytoplasm (within the cell membrane).

• Proteins formed from free ribosomes are used within the cell.

• Membrane-bound ribosomes place newly produced polypeptides directly into the endoplasmic reticulum.

• Usually produce proteins that are used within the cell membrane or are expelled from the cell via exocytosis.
• Proteins containing disulfide bonds using cysteine cannot be produced outside of the lumen of the endoplasmic reticulum.

Composed of 60% ribosomal RNA and 30% ribosomal proteins (known as a Ribonucleoprotein or RNP).

• Composed of 60% ribosomal RNA and 30% ribosomal proteins (known as a Ribonucleoprotein or RNP).

Crystallographic work has shown that there are no ribosomal proteins close to the reaction site for polypeptide synthesis.

• Crystallographic work has shown that there are no ribosomal proteins close to the reaction site for polypeptide synthesis.

• This suggests that the protein components of ribosomes act as a scaffold that may enhance the ability of rRNA to synthesize protein rather than directly participating in catalysis.

Their active sites (A, P & E) are made of RNA, so ribosomes are now classified as "ribozymes".

• Their active sites (A, P & E) are made of RNA, so ribosomes are now classified as "ribozymes".

The ribosome has three binding sites for tRNA molecules: the A, P and E sites.

• The ribosome has three binding sites for tRNA molecules: the A, P and E sites.

• A= aminoacyl-tRNA site
• P= Peptidyl-tRNA site
• E= Egress site

The ribosome has three binding sites for tRNA molecules: the A, P and E sites.

• The ribosome has three binding sites for tRNA molecules: the A, P and E sites.

• A= aminoacyl-tRNA site
• P= Peptidyl-tRNA site
• E= Egress site

The ribosome has three binding sites for tRNA molecules: the A, P and E sites.

• The ribosome has three binding sites for tRNA molecules: the A, P and E sites.

• A= aminoacyl-tRNA site
• P= Peptidyl-tRNA site
• E= Egress site

The ribosome has three binding sites for tRNA molecules: the A, P and E sites.

• The ribosome has three binding sites for tRNA molecules: the A, P and E sites.

• A= aminoacyl-tRNA site
• P= Peptidyl-tRNA site
• E= Egress site

The ribosome has three binding sites for tRNA molecules: the A, P and E sites.

• The ribosome has three binding sites for tRNA molecules: the A, P and E sites.

• A= aminoacyl-tRNA site
• P= Peptidyl-tRNA site
• E= Egress site

Their active sites (A, P & E) are made of RNA, so ribosomes are now classified as "ribozymes".

• Their active sites (A, P & E) are made of RNA, so ribosomes are now classified as "ribozymes".

Prokaryote and eukaryote ribsome differences lie outside of functional parts (A, P & E sites) and therefore “redundant insertions”.

• Prokaryote and eukaryote ribsome differences lie outside of functional parts (A, P & E sites) and therefore “redundant insertions”.

• The differences are exploited by pharmaceuticals to create antibiotics that destroy a bacterial without harming the cells of the infected person.

• Even though mitochondria possess similar ribosomes they are not affected by these antibiotics (double membrane).

• Antibiotics such as macrolides, aminoglycosides and others:

Structure of the antibiotic gentamicin C1a bound to its rRNA target.

• Structure of the antibiotic gentamicin C1a bound to its rRNA target.

• Gentamicin, an aminoglycoside antibiotic, binds within the major groove of the RNA, which is located in the decoding site of the bacterial ribosome.

• Aminoglycosides cause misreading of the genetic code.

• Binding of the drug causes a conformational change in ribosomal RNA that disrupts high-fidelity reading of the genetic code.

Structure of the aminoglycoside paromomycin bound to the bacterial rRNA decoding site.

• Structure of the aminoglycoside paromomycin bound to the bacterial rRNA decoding site.

• The sites that lead to resistance are highlighted with purple spheres.

• The N7 methylation at G1405 only causes resistance to aminoglycosides like gentamicin that contact this position directly.

Ribosomes are the workhorse for protein biosythesis= TRANSLATION

• Ribosomes are the workhorse for protein biosythesis= TRANSLATION