Biol 141 Class 10

Central Dogma

Transcription and Translation (Prokaryotes v. Eukaryotes)

Bacteria (Prokaryotes)

• All (transcription and translation) happen in the cytoplasm

Eukaryotes

• Transcription in nucleus

• Translation in cytoplasm

RNA Polymerase

The enzyme that makes RNA

• Takes information from DNA to build mRNA

• It is an enzyme made out of proteins

• Synthesizes RNA from DNA template (Transcription)

A sequence indicates the end of RNA polymerase

• When done the mRNA “falls off”

Bacteria

• Has 1 RNA polymerase

Eukaryotes

• Have 3 RNA polymerase:

• RNA po I- transcribes ribosomal RNAs

• RNA pol II- transcribes mRNA for protein-coding genes

• RNA pol III- transcribes tRNAs

Transcription: Bacteria (Initiation)

1. Promoter

2. Sigma Protein

3. RNA polymerase

Bacteria have several different sigma proteins

Promoter (Bacteria)

Specific sequence on DNA that signals where transcription should start

• Promotes NOT transcribed

• Indicate RNA polymerase where to stop, initiates transcription

Sigma Protein (Bacteria)

Sigma protein binds to the promoter (Different factors in a eukaryotes)

• Indicate to RNA polymerase where they should start 

• Binds to the RNA polymerase and then guides it to the promoter so that transcription can occur 

Transcription Initiation (Eukaryotes)

Use transcription factors bind to promoter

• TATA Box- A short DNA sequence in a eukaryotic promoter that acts like a “start signal’ for RNA polymerase to begin transcription

Transcription: Elongation (Prokaryotes & Eukaryotes)

1. Building RNA Strand

2. Phosphodiester Linkage

Building RNA strand

DNA

• DNA has 2 strands (forms helix)

• The strand that is not used for transcription is the non template strand

• The strand that is used for transcription (RNA polymerase attaches to it) is the template strand

Hydrogen bonds form between complementary base pairs 

• Between DNA template and RNA

• In DNA, there are hydrogen bonds between the base pairs that allow DNA to be double stranded

• During transcription this helix is broken

• As RNA is being created it forms temporary hydrogen bonds with DNA

• When RNA is done making complementary base pairs of DNA it detaches from it

Phosphodiester Linkage

Backbone of RNA

• Linkage is formed by RNA polymerase after base pairing occurs

• The phosphate of one nucleotide connects to the sugar of the next nucleotide (sugar→ phosphate → sugar…)

DIFFERENCE- RNA poly in eukaryotes needs transcription factors instead of sigma factors

Energy to build RNA strand

When the RNA polymerase adds a nucleoside triphosphate (A, U, C, G) to growing RNA strand it breaks off two phosphates releasing energy 

• The RNA polymerase finds nucleotides that align with DNA

• It then adds these monomers/ nucleoside triphosphate to growing RNA strand

• When bonded, two phosphates break off

• This breaking of bonds releases energy (chemical bonds rules don’t apply because reaction favors energy release)

• This is where RNA polymerase gets it energy to build RNA strand

RNA polymerase reading code

RNA polymerase ‘reads’ the template in the reverse manner 3’ → 5’ but polymerizes the RNA 5’ → 3’

• The RNA polymerase start at the 3’ of DNA and goes to 5’

• It does this so that its strand can read correctly from 5’ to 3’ 

5’ Cap and 3’ Poly A tail (Eukaryotes only)

Have a 5’ CAP and a 3’ polyA tail

• Only in eukaryotes

5’ CAP 

• Helps RNA bind to ribosomes for translation

• Guides RNA strand to ribosome

• As soon as the RNA emerges from RNA polymerase, a modified guanine nucleotide is added to its 5’ end (5’ cap)

3’ PolyA tail

• Protects RNA from degradation 

• Helps push RNA strand out of nucleus

• After transcription is done a poly A tail is added to the 3’ end  of RNA 

Splicing and Heteroduplex

Splicing

• Removes introns (non-coding parts), leaves in exons (coding parts), carried out by a ribozyme

• Alternative splicing- can vary to change the protein created, essentially different splicing codes for different proteins

• Certain DNA sequence does not get coded for by the RNA polymerase (this helps make different proteins from same sequence)

• ONLY IN EUKARYOTES

To create RNA, DNA has to be single-stranded

• The single-stranded DNA base paired (pairs) with mRNA 

• They are heteroduplex- different types of nucleic acids (two strand come from different sources)

• DNA is originally double stranded but has to opened, causing the hydrogen bonds between those two strands to break

• RNA temporarily becomes DNA’s other strand

Protein Synthesis of Eukaryotes

DNA splits apart and goes through the process of transcription to create RNA

RNA is then taken to the cytoplasm (which contains ribosomes) and here it is translated into a protein

Introns v Exons

Gene

• Section of DNA with the information to construct protein

Transcription

• Process of using DNA template to create a strand of RNA

• Introns- non-coding part

• Exon- coding part, expressed

• Both introns and exons are transcribed

Introns and Exons in RNA

• Introns are going to be removed from the strand of pre- RNA (broken down and recycled)

• Exons are spliced with other exons into a long chain of mature RNA

Transcription: Termination (Prokaryotes)

When bacteria is done transcribing, the sequence in RNA that will form a hairpin loop causing RNA pol to come off

• Fold into U until an enzyme causes it to fall of

• RNA contains palindromic, a sequence that can fold back and pair with itself

• When RNA wants to break off of DNA it forms a U shape so that the RNA polymerase and the RNA strand are detached 

Transcription: Termination (Eukaryotes)

Sequence of polyAs in mRNA is a recognition site for an enzyme to cut/release the mRNA (This is NOT the 3’- poly A tail)

• There is a sequence in DNA that codes for the termination of RNA polymerase

• This signals the detachment of the RNA strand from DNA, enzymes (Xrn2) help RNA polymerase detach

Both Eukaryotes & Prokaryotes have STOP SEQUENCES for TRANSCRIPTION

Translation (Eukaryote v Bacteria)

Bacteria

• All happens in the cytoplasm, so translation can start before transcription is complete 

Eukaryote

• Pre-mRNA processed (CAP, tail & splicing) into mature mRNA in nucleus, then exported to the cytoplasm

Translation (tRNA)

Translation occurs in ribosomes which are in the cytoplasm

• Ribosomes has 2 subunits (1 large and 1 small subunit)

• It has A, P, and E site 

Peptide bond of amino acids are form

• The folding of this peptide bond (primary, secondary, tertiary, quaternary) determines the function of the protein

A- Site 

Arrival of amino acid

• Each codon (3 nucleotides) code for a specific amino acid

• Each tRNA has 3 nucleotide sequences (anticodon)

• A tRNA arrives to the A site and if its anticodon matches the codons of the RNA strand (base-pairing rules) they will bond (tRNA and RNA strand)

• Once these molecules bind, it changes its shape and it moves to the P-site

P-Site

Peptide (protein) is going to stay

• Since the first 3 bases (codons) of the mRNA have been attached to their anticodon, which is on tRNA, it is moved to the P-site

• This makes space for another arrival of tRNA at the A-site

• The first amino acids (in the P-site) forms a peptide bonds with the new amino acid (in the A-site)

• After bonding the second amino acid (in A-site) moves to the P-site and the amino acid in the P-site moves to the E-site, however it leaves behind the bonded amino acid 

Translation BEGINS IN THE P-SITE

E-Site

Exit Site

• Since the amino acid of the tRNA has been left in a peptide bond with the other amino acid in the P-site, it no longer serves a purpose

• tRNA leaves the ribosome (without amino acid)

• This opens up space for the other tRNA molecule in the P-site to move to the E-site

Codon

For every 3 nitrogen bases 1 codon is produced

• 3 nitrogen bases= codon- → 1AA

• Every codon codes for 1 amino acid

There are 64 different codons (triplets), but there are only 20 different amino acids

• Multiple codons code for same amino acid

• Genetic code is degenerate- more than one triplet may code for an amino acid

AUG is the starting codon

Translation: Initiation in Bacteria

Initiation Factors bind to ribosome-binding site (RBS), they help mRNA bind to small subunit and tRNA bind to correct site in the large subunit

• They align AUG start codon in P-site of ribosome, anticodon on tRNA is UAC

• f-met becomes the first amino acid translated, 1st amino acid

• The first tRNA is always a tRNA^met (tRNA with the amino acid methane attached), which binds to AUG

• Signaling ribosome assembly (start)

Translation: Initiation (Eukaryotes)

Initiation Factors bind to ribosome-binding site (RBS) and 5’ CAP, help set up translation process

• Align AUG start codon of mRNA strand in P-site of ribosome

• Met becomes the first amino acid translated

• Signaling ribosome assembly (start)

TRANSLATION INITIATION SIMILAR IN BOTH PROKARYOTES & EUKARYOTES

Molecular Players (Translation)

tRNA Synthetases

• Enzymes that add correct amino acid onto specific tRNAs

tRNA

• RNA molecules with 3d structures (clover) that have an anticodon at one end which will base-pair to a codon, and an amino acid attached at the other

• tRNA is in the cytoplasm, the right tRNA (carrying the right aa) will bind to the the codon of the pre-mRNA

Ribosome

Ribosome 

Huge protein-RNA complex that links amino acids by peptide bonds, translating an RNA message into a peptide (protein)

• Made in the nucleus 

  • Made of ribosomal RNA

  • Has 2 subunits- One large, the other small 

  • Form ‘snowman’

  • It has E, P, and A site

  • Translate anticodon of RNA to form proteins

Translation Termination (Prokaryotes vs. Eukaryotes) 

Prokaryotes

• A stop codon is reached

• Stop codons= UAA, UAG, UGA, no amino acid

• Release factors (special proteins) come in and help the ribosome release the finished protein

• The ribosome, mRNA, and tRNA separate

Eukaryotes

• tRNA with no amino acid is reached (stop codon), signals the end of the mRNA translation (no amino acid= no more growing peptide bond)

• There is no amino acid for the termination of translation

Proteinaceous Release Factor = All components come apart (subunits of ribosome no longer need to be assembled)

Translation Process

Initiation

1. mRNA binds to small subunit [with help of initiation factors]

2. Initiator aminoacyl tRNA (UAC) binds to start codon (AUG)

3. Large subunit of ribosome binds, completing ribosome assembly

Elongation

1. Incoming aminoacyl tRNA (in A-site)

2. Peptide bond formation

3. Translocation (to E-site)

4. Repeat

Termination

1. Release factors binds to stop codons

2. Polypeptide and uncharged tRNA (no AA) are released

3. Ribosome subunits separate