IX: DNA Function: Protein Synthesis. A. Overview: 1. The central dogma of genetics: unidirectional...

IX: DNA Function: Protein Synthesis


A. Overview:

1. The central dogma of genetics: unidirectional flow of information


A. Overview:


2. Why a two-step process?

a. Historical contingency…That’s how it evolved from an RNA Protein system…


A. Overview:



- it evolved that way….

- because it is more productive….

tRNA


A. Overview:


2. The code is:


4. Players:

ds-DNA; GENE (recipe)

RNA polymerases make:m-RNA (gene transcript)r-RNA (reader in ribosome)t-RNA (AA carrier)

tRNA


A. Overview: B. Transcription:



1. The DNA template:

In a given region (gene), only one strand is transcribed; only one strand carries a message that makes ‘sense’. The sequence on the other strand is limited to being complementary to the first strand.

3’ 5’

3’5’

‘sense’ strand

…C A T…

‘anti-sense’ strand

…G T A…





There is a region ‘upstream’ from the gene called the PROMOTER. This is where the RNA Polymerase binds. The polymerase is attracted to particular sequences. Many are consensus sequences found upstream from different genes, and across many many species.

3’ 5’

3’5’

‘sense’ strand

…C A T…


…G T A…





There is a region ‘upstream’ from the gene called the PROMOTER. This is where the RNA Polymerase binds. The polymerase is attracted to particular sequences. Many are consensus sequences found upstream from different genes, and across many many species. In all bacteria, the sequence TATAAT lies 10 bases upstream from all bacterial genes, and TTGACA lies 35 bases upstream. Two binding sites create a directionality.

Promoters can be 40 bases long. Frequency of binding is affected by variation in the rest of the sequence….

3’ 5’

3’5’

‘sense’ strand

…C A T…


…G T A…





In all eukaryotes, there is a consensus sequence of TATA in all promoters at -35, and a CAAT box at -80. There are also enhancer regions that can modulate binding, and Transcription Factors that bind to the promoter and increase/decrease the efficacy of polymerase binding.

3’ 5’

3’5’

‘sense’ strand

…C A T…


…G T A…





In all eukaryotes, there is a consensus sequence of TATA in all promoters at -35, and a CAAT box at -80. There are also enhancer regions that can modulate binding, and Transcription Factors that bind to the promoter and increase/decrease the efficacy of polymerase binding.

Why is there consensus in promoter sequences across all life? What does that say about how well mutations are tolerated in these regions?

3’ 5’

3’5’

‘sense’ strand

…C A T…


…G T A…





There is region downstream called a TERMINATOR (40 bases long).

3’ 5’

3’5’

‘sense’ strand

…C A T…


…G T A…





So, the polymerase binds at the promoter, transcribes the whole gene, and decouples at the terminator.

3’ 5’

3’5’

‘sense’ strand

…C A T…


…G T A…





In bacteria, the gene is a continuous coding sequence….

3’ 5’

3’5’

‘sense’ strand


Continuous recipe for a protein






In eukaryotes, genes contain non-coding, intervening sequences called introns; the coding sequences are called exons.

3’ 5’

3’5’

‘sense’ strand


Disconti nuous recipe for a protein






In eukaryotes, genes contain non-coding, intervening sequences called introns; the coding sequences are called exons. Although transcription is continuous and every base is transcribed, RNA processing is required to splice out the non-coding introns and create a continuous reading frame for translation.

3’ 5’

3’5’

‘sense’ strand


Disconti nuous recipe for a protein

There are two alternate hypotheses for the evolution of introns:

Eubacteria (no introns at all)

Archaea (introns in r-RNA and t-RNA genes) Eukarya (introns)


“Introns Early”: The ancestral structure was a split gene structure, favored because evolution could proceed rapidly by the shuffling of functional exons to create new genes (exon shuffling hypothesis).

Eukarya (introns)Eubacteria (no introns at all)

Archaea (introns in r-RNA and t-RNA genes)


“Introns Early”: The ancestral structure was a split gene structure, favored because evolution could proceed rapidly by the shuffling of functional exons to create new genes (exon shuffling hypothesis).

Eukarya (introns)

Introns were lost from prokaryotes because of the extreme selective advantage for rapid division.

Eubacteria (no introns at all)



“Introns Late”: The ancestral structure was a continuous gene structure. Introns evolved as “transposeable elements” and inserted themselves and multiplied only in eukaryotic ancestors.

Eukarya (introns)Eubacteria (no introns at all)


The split-gene structure of eukaryotes was discovered in 1977:

Philip Sharp and coworkers found that viral genes in eukaryotes and initial transcripts were longer than the functional m-RNA or proteins.

The split-gene structure of eukaryotes was discovered in 1977:

Philip Sharp and coworkers found that viral genes in eukaryotes and initial transcripts were longer than the functional m-RNA or proteins.

1978: Walter Gilbert coins the terms ‘intron’ and ‘exon’.

Heteroduplex analyses of DNA and m-RNA show “loops” of RNA that have no complement in the DNA template strand:

All eukaryotic genes except those coding for histones have introns; some make up the vast majority of the ‘gene’, itself:



1. The DNA template:2. RNA Polymerase:

In bacteria, there is only one enzyme; consisting of a ‘core enzyme’ (responsible for polymerization), and subunits that affect different functions. For example, the sigma subunit is responsible for initiation, and the rho subunit stimulates termination.

3’ 5’

3’5’

‘sense’ strand

…C A T…


…G T A…




In bacteria, there is only one enzyme; consisting of a ‘core enzyme’ (responsible for polymerization) with two polypeptides, and subunits that affect different functions. For example, the sigma subunit is responsible for initiation, and the rho subunit stimulates termination. There are different sigma subunits that affect polymerase binding; complementing the variations in promoters.

3’ 5’

3’5’

‘sense’ strand

…C A T…


…G T A…




In bacteria, there is only one enzyme; consisting of a ‘core enzyme’ (responsible for polymerization) with two polypeptides, and subunits that affect different functions

In eukaryotes, there are three RNA Polymerases:

3’ 5’

3’5’

‘sense’ strand

…C A T…


…G T A…



1. The DNA template:2. RNA Polymerase:3. RNA triphosphate precursors:

3’ 5’

3’5’

‘sense’ strand

…C A T…


…G T A…

A U

U

A



1. The DNA template:2. RNA Polymerase:3. RNA triphosphate precursors:4. Process in Bacteria

3’ 5’

3’5’

‘sense’ strand

…C A T…


…G T A…




a. Polymerase (with sigma) lands on ds-DNA at promoter

3’ 5’

3’5’

‘sense’ strand

…C A T…


…G T A…




a. Polymerase (with sigma) lands on ds-DNA at promoterb. Helicases separate strands; first bases linked 5’ 3’

3’ 5’

3’

5’

‘sense’ strand

C C C A G T C A T G G G T….

‘anti

-sen

se’ s

tran

d

G G G5’ 3’




a. Polymerase (with sigma) lands on ds-DNA at promoterb. Helicases separate strands; first bases linked 5’ 3’c. Sigma subunit dissociates, polymerization continues

3’ 5’

3’

5’

‘sense’ strand

C C C A G T C A T G G G T….

‘anti

-sen

se’ s

tran

d

G G G UC A GU A C C C A…5’ 3’




a. Polymerase (with sigma) lands on ds-DNA at promoterb. Helicases separate strands; first bases linked 5’ 3’c. Sigma subunit dissociates, polymerization continuesd. Termination: sequences rich in C’s and G’s, followed by A’s and T’s

3’ 5’

3’

‘sense’ strand

C C C C G C A A G C G G G G A A T T….G G G GC G UU C G C C C C U U A A…

5’ 3’




a. Polymerase (with sigma) lands on ds-DNA at promoterb. Helicases separate strands; first bases linked 5’ 3’c. Sigma subunit dissociates, polymerization continuesd. Termination: sequences rich in C’s and G’s, followed by A’s and T’s - Rho Independent:

- the C’s and G’s in the m-RNA for a ‘stem-loop’ structure; and binds to a protein bound to the Polymerase (nusA)

3’ 5’

3’

‘sense’ strand

C C C C G C A A G C G G G G A A T T….

G G

G G

C G U

5’ 3’

U C

G C

C C

C





- the C’s and G’s in the m-RNA for a ‘stem-loop’ structure; and binds to a protein bound to the Polymerase (nusA) - this causes the polymerase to pause, just as it is reading

the area rich in A’s… which have fewer h-bonds. The pausing and the destabilization of the polymerase caused by the ‘stem-loop’ causes the m-RNA/polymerase to detach.

3’ 5’

3’

‘sense’ strand


G G

G G

C G U

5’ 3’

U C

G C

C C

C





- recent research has identified certain RNA’s that loop and bind a small protein produced BY their own code. So, if the product concentration is high, the m-RNA binds the protein, loops, and shuts down transcription (down-regulating the gene). These RNA’s that turn off their own gene are “riboswitches”

3’ 5’

3’

‘sense’ strand


G G

G G

C G U

5’ 3’

U C

G C

C C

C




a. Polymerase (with sigma) lands on ds-DNA at promoterb. Helicases separate strands; first bases linked 5’ 3’c. Sigma subunit dissociates, polymerization continuesd. Termination: sequences rich in C’s and G’s, followed by A’s and T’s - Rho-dependent:

Rho – a circular hexamer, binds the m-RNA in C-G rich regions

3’ 5’

3’

‘sense’ strand


5’ 3’




a. Polymerase (with sigma) lands on ds-DNA at promoterb. Helicases separate strands; first bases linked 5’ 3’c. Sigma subunit dissociates, polymerization continuesd. Termination: sequences rich in C’s and G’s, followed by A’s and T’s - Rho-dependent:

Rho – a circular hexamer, binds the m-RNA in C-G rich regions it slides up the strand; decoupling the polymerase from the m-RNA at A-T rich sites.

3’ 5’

3’

‘sense’ strand


5’ 3’




a. Polymerase (with sigma) lands on ds-DNA at promoterb. Helicases separate strands; first bases linked 5’ 3’c. Sigma subunit dissociates, polymerization continuesd. Termination:e. Polycistronic DNA:

In bacteria, proteins involved in the same metabolic process are often encoded by neighboring genes that are read as a UNIT (operon),

Three genes ‘read’ as a single unit





In bacteria, proteins involved in the same metabolic process are often encoded by neighboring genes that are read as a UNIT (operon), producing one m-RNA that has the transcript of all three genes…






In bacteria, proteins involved in the same metabolic process are often encoded by neighboring genes that are read as a UNIT (operon), producing one m-RNA that has the transcript of all three genes… then, in TRANSLATION, ribosomes attach at ‘start codons’ along the strand, synthesizing all proteins simultaneously.




1. The DNA template:2. RNA Polymerase:3. RNA triphosphate precursors:4. Process in Bacteria:Translation of m-RNA by ribosomes occurs even before m-RNA is complete!



1. The DNA template:2. RNA Polymerase:3. RNA triphosphate precursors:4. Process in Bacteria:5. Process in Eukaryotes:




- The chromatin must be unwound = chromatin remodeling




- The chromatin must be unwound = chromatin remodeling - Initiation is regulated by ‘enhancer sequences’ upstream and

downstream from the gene, and transcription factor binding.





downstream from the gene, and transcription factor binding.

The enhancer sequences are “cis-acting regulatory elements” (CRE’s), because they are on the same chromosome as the gene. Since the transcription factors are proteins encoded elsewhere in the genome, even on different chromosomes, they are called “trans-acting regulatory elements”.





downstream from the gene, and transcription factor binding. - Because the m-RNA is bound in the nucleus—separated from the

ribosomes—translation does not take place immediately.





downstream from the gene, and transcription factor binding. - Because the m-RNA is bound in the nucleus—separated from the

ribosomes—translation does not take place immediately. - Most significantly, the initial RNA product is PROCESSED.


A. Overview: B. Transcription: C. RNA Processing:



- In prokaryotes, only r-RNA genes have introns; protein-encoding genes have a continuous coding sequence.



- In prokaryotes, only r-RNA genes have introns; protein-encoding genes have a continuous coding sequence. As such, the m-RNA can be translated as soon as it is made.



- In eukaryotes, the initial RNA transcripts contain introns that are spliced out.




- In addition a 7-mG cap and poly-A tail are added to the processed RNA; probably to reduce the rate of exonuclease activity in the cytoplasm.




- In addition a 7-mG cap and poly-A tail are added to the processed RNA; probably to reduce the rate of exonuclease activity in the cytoplasm).

- Group I introns splice themselves out of r-RNA; they have auto-catalytic function. These were the first RNA molecules found that had enzyme-like catalytic properties = ribozymes (Cech, 1982)

Free guanine nucleoside



- Group I introns splice themselves out of r-RNA; they have auto-catalytic function. These were the first RNA molecules found that had enzyme-like catalytic properties = ribozymes (Cech, 1982)

- Group II introns are autocatalytic, too, and occur in mitochondria and chloroplast m,t-RNA.



- Group I introns - Group II introns- nuclear introns:

m-RNA transcripts in the nucleus are very large, and processing is more complicated




m-RNA transcripts in the nucleus are very large, and processing is more complicated. Terminal ‘GU-AG’ sequences are recognized by snRP’s (‘snurps’) that have sn-RNA’s rich in Uracil (‘U’ designations).

sn-RNA = short, nuclear RNA… also “U-RNA”snRP’s = short, nuclear, riboproteins




m-RNA transcripts in the nucleus are very large, and processing is more complicated. Terminal ‘GU-AG’ sequences are recognized by snRNP’s (‘snurps’) that have sn-RNA’s rich in Uracil (‘U’ designations). Binding of complementary snRP’s creates the spliceosome, which creates a lariat structure in the RNA.




m-RNA transcripts in the nucleus are very large, and processing is more complicated. Terminal ‘GU-AG’ sequences are recognized by snRNP’s (‘snurps’) that have sn-RNA’s rich in Uracil (‘U’ designations). Binding of complementary snRP’s creates the spliceosome, which creates a lariat structure in the RNA. The intron is cleaved, and the exons are ligated together.




m-RNA transcripts in the nucleus are very large, and processing is more complicated. Terminal ‘GU-AG’ sequences are recognized by snRNP’s (‘snurps’) that have sn-RNA’s rich in Uracil (‘U’ designations). Binding of complementary snRPS’s creates the spliceosome, which creates a lariat structure in the RNA. The intron is cleaved, and the exons are ligated together. This splicing can vary, such that a single m-RNA can be spliced at different places and produce different proteins…ultimately from the same gene!



So, the final product has a 7mG cap, poly-A tail, and a continuous message.

This ‘mature’ m-RNA leaves the nucleus and enters the cytoplasm, where it will be translated.

IX: DNA Function: Protein Synthesis. A. Overview: 1. The central dogma of genetics: unidirectional...

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Transcript of IX: DNA Function: Protein Synthesis. A. Overview: 1. The central dogma of genetics: unidirectional...