Transcription Flashcards
Lecture Outcomes
Describe the structure of RNA and the differences to DNA
- Define the term transcription, understand how DNA is transcribed to RNA
- List the differences in the transcription processes between eukaryotes
and prokaryotes - Describe and understand the function of RNA polymerase
- Describe the transcription initiation and termination signals in DNA
- Describe the process of capping and polyadenylation
- List the different classes of RNA molecules
The Central Dogma photo
Structure of RNA photo
The Central Dogma
- Portions of DNA sequence are copied into RNA
- Genes
- Intergenic spaces can also be transcribed
- Eukaryotic and Prokaryotic RNA is
processed differently - Not all RNA is translated, only m(essanger)
RNA directs protein synthesis
Transcription: Requirements
- Definition of “TRANSCRIPTION”
– “The production of RNA from a DNA template”
- RNA - is made in a manner similar to DNA replication but only one strand is copied.
- Enzymatic synthesis of RNA requires:
- The four ribonucleoside 5’ triphosphates
5’ATP 5’GTP 5’CTP 5’UTP
- Mg2+
- DNA template (no primer required)
- RNA polymerase (RNAP)
Mechanisms of synthesis: initiation, elongation and termination
RNA polymerase
- Similar to DNA polymerase
– Forms phosphodiester bonds between ribonucleosides
– Uses energy stored in ribonucleoside triphosphates for polymerisation
- Unlike DNA polymerase
– RNA synthesis starts without a primer
– Error rate higher than DNA polymerase
– Unwinding of the DNA template does not require a helicase or ATP.
Transcription: conventions
- DNA sequences are written in the 5’ to 3’ direction
- The “coding” or “sense” DNA strand is written in the 5’-3’ direction
- RNA polymerase makes a complementary copy of the anti-parallel strand (template) in the
3’ to 5’ direction from the promoter (P) to the terminator (T) - Transcribed mRNA is written in the 5’ to 3’ direction and is the same sequence as the coding strand in the DNA except that U is substituted for T.
Overall reaction photo
XTP represents the first nucleotide at the 5’ terminus of the RNA chain
NMP is a mononucleotide in the RNA chain,
RNA-P is RNA polymerase
PPi is the pyrophosphate released each time a nucleotide is added to the growing chain.
The Mg2+ ion is required for all nucleic acid polymerization reactions.
RNA polymerase photo
E. coli RNA polymerase
- E. coli RNA polymerase has 5 SUBUNITS (2 x α, β, β’ and ω). The complex of α2ββ’ ω is called the CORE ENZYME
- σ is responsible for promoter recognition (pyridine-rich DNA sequence of 10 or more bases). Associates with core enzyme to form the HOLOENZYME.
- Once correct initiation has been achieved, σ dissociates from the HOLOENZYME and the core enzyme continues elongation of the RNA chain.
E. coli RNA polymerase photo
Transcription in Prokaryotes photo
Transcription initiation and termination signals in DNA photo
Transcription termination signals in
E. coli
Can be achieved by one of two mechanisms
- DNA sequence dependant: an INVERTED REPEAT followed by a stretch of T bases
- DNA sequence dependent: bound to a protein factor (Rho)
Transcription Initiation signals in E. coli
Transcription signals in E. coli
In both cases the core enzyme associates and interacts with a free σ subunit and reforms the HOLOENZYME, which may then initiate further transcription (recycles RNA polymerase)
Transcription termination signals in
E. coli photo
Transcription signals in E. coli photo
Prokaryotic messenger RNA
- mRNA molecules are a copy of the coding strand of DNA sequences which determine the amino acid sequence of all proteins
- A DNA segment corresponding to one polypeptide chain plus the
start and stop signals is called a CISTRON. A single mRNA
encoding a single polypeptide is called MONOCISTRONIC mRNA - In prokaryotes POLYCISTRONIC mRNAs are quite common and
code for several different polypeptide chains - mRNA’s have 5’ LEADERS, 3’ TERMINI and, in the case of
polycistronic mRNAs, they have INTERCISTRONIC regions called
SPACERS - Prokaryotic mRNA’s have short half lives, generally a few minutes
only - Primary transcript not processed
Prokaryotic messenger RNA photo
Eukaryotic messenger RNA
- Gene contains coding (exons) and non-coding (introns) regions
- RNA is processed
– RNA capping (G with methyl group added to 5’ end)
– Polyadenylation (series of “A” added to 3’ end)
– Identifies RNA molecule as an mRNA
- Introns removed by a splicesome which recognises the boundaries between introns and exons
- Exons stitched back together and translated
- mRNA molecules eventually degraded
Eukaryotic messenger RNA photo
Classes of RNA molecules
Lecture Outcomes
- Describe the major classes of RNA molecules
- Describe the differences between prokaryotic and eukaryotic mRNA
- Describe the structure and function of tRNA and function of aminoacyl-tRNA synthetases
- Describe differences between prokaryotic and eukaryotic ribosomes
- Understand the roles of ribosome binding sites in translation of mRNA to protein
- Learn the definitions of polycistronic mRNA, polyribosome, mRNA cap, poly-A tail, codon, anticodon, amino acyl tRNA
There are many steps between DNA
and Protein
The production of a protein by a
eukaryotic cell.
The final level of each protein in a cell depends on the efficiency of each step.
There are many steps between DNA
and Protein photo
Three major classes of RNA molecules
- Messenger RNA (mRNA):
- copy of DNA which codes for the protein
- Transfer RNA (tRNA):
- tRNA’s are small adaptor molecules which align specific amino acids opposite their triplet CODON in the mRNA molecule during the translation of mRNA into protein
- Ribosomal RNA (rRNA):
- rRNA is an integral part of ribosomal structures and is an important part of the protein synthesising machinery
These 3 interact to enable translation of the mRNA to protein
Eukaryotic and Prokaryotic cells handle
their RNA transcripts differently
Eukaryotes
- Transcription/translation
occur separately - In different compartments
- Enables greater control
Prokaryotes
- Transcription/translation
are coupled - Occur simultaneously
Eukaryotic and Prokaryotic cells handle
their RNA transcripts differently
Unlike bacterial mRNAs, eukaryotic mRNAs are
modified by capping and polyadenylation
- Cap = 7-methylguanosine
- Poly-A tail = long string of adenosines linked to 3’-end of mRNA:
- Not incorporated in the DNA sequence
- Binding site for several proteins
- Roles of 5’ end cap and 3’ end polyadenylation:
- Assist the export of mRNA molecules from the nucleus
- Protect the mRNA from degradation in the cytoplasm and increase its half life
- Enable the binding of ribosomes, promote translation
mRNA sequence is decoded in sets of 3 nucleotides called codons photo
Transfer RNAs
- Amino acid cannot directly recognise the mRNA codon, needs
adaptor. - tRNAs are small adaptor molecules ~ 80 nucleotides long:
- align specific amino acids opposite their triplet codon in the mRNA molecule during translation
- Bases other than A, G, C and U are present in tRNA as a result of
post-transcriptional modification e.g. inosine (a modified adenine), pseudouridine, dihydrouridine
tRNA Structure photo
Genetic code is translated by two
adaptors
Acts 1st : Aminoacyl-tRNA synthetase couples a particular amino acid to its corresponding tRNA
Acts 2nd : tRNA whose anticodon forms base pairs with codon of mRNA
tRNA Structure
- Internal complementary base pairing of RNA gives cloverleaf structure
- Anticodon sequence of 3 bases determines mRNA codon binding
- Amino acid attachment site is at 3’ end of tRNA
- Modified bases
- D = dihydrouracil
- Ψ = pseudouridine
- 3D shape determines attachment of correct amino acid (matching the codon/anticodon pair) by aminoacyl-tRNA synthetases
tRNA Structure photo
Genetic code is translated by two
adaptors photo
Aminoacyl-tRNA Synthetases
- For every amino acid one aminoacyl-tRNA synthetase exists:
i.e. links a particular amino acid to several tRNAs
- These enzymes are responsible for the attachment of the correct amino acid to the tRNA:
i.e. the amino acid specified by the anticodon of the tRNA and the amino acid attachment sequence on the 3’ end
Terminology:
- leucyl-tRNA synthetase attaches leucine (aa) to tRNALeu
- alanyl-tRNA synthetase attaches alanine to tRNAAla, and etc.
- When the correct amino acid has been attached to its tRNA, the tRNA is said to be CHARGED or ACYLATED
- Termed an amino acyl tRNA
- ATP hydrolysis required
- mischarging means an incorrect amino acid attachment
Aminoacyl-tRNA Synthetases photo
Prokaryotic Ribosome structure photo
Prokaryotic Ribosome structure
- rRNAs are an integral part of ribosomal structures and are important parts of the protein synthesising machinery
- Prokaryotes have 5S, 23S rRNA in their large ribosomal subunit
- Prokaryotes have 16S rRNA in their small ribosomal subunit
Ribosomal RNAs
The mRNA message is decoded on ribosomes
- The ribosome is a very large multi-domain complex
- It scans along the mRNA and captures the correct aminoacyl tRNA:
- Correct complementarity (base-pairing) results in the covalent linking of aa onto the growing polypeptide chain
Ribosomes in the cytoplasm of a eukaryotic cell. Electron micrograph shows ribosomes as black dots (arrows).
Some are free, some attached to endoplasmic reticulum.
Eukaryotic Ribosome structure
- Eukaryotes have 5S, 5.8S and 28S rRNA in their large ribosomal subunit
- Eukaryotes have 18S rRNA in their small ribosomal subunit
- Despite differences in protein and rRNA components, both prokaryotic and eukaryotic ribosomes have nearly the same structure and function
Elongation of
polypeptide photo
Ribosomes have binding sites
- Small subunit matches tRNA to the codon on the mRNA
- Large subunit catalyzes the formation of the peptide bonds that link the amino acids together into a polypeptide
- Each ribosome has one binding site for mRNA and three binding sites for tRNA tRNA binding sites:
A = aminoacyl-tRNA : new tRNA enters ribosomal complex
P = peptidyl-tRNA : tRNA attached to the polypeptide chain
E = exit : empty tRNA exits ribosomal complex
Eukaryotic Ribosome structure photo
Ribosomes have binding sites photo
A single prokaryotic mRNA molecule can encode several different proteins
- Unlike eukaryotic ribosomes, which recognize a 5’ cap, prokaryotic ribosomes initiate translation at ribosome-binding sites, which can be located in the interior of an mRNA molecule
- This feature permits prokaryotes to synthesize more than one type of protein from a single polycistronic mRNA molecule
A single prokaryotic mRNA molecule can encode several different proteins photo
Proteins are translated by polyribosomes
- Many ribosomes can simultaneously translate the same mRNA molecule
- Increases the production capacity of a particular protein
polyribosomes photo
