L4 RNA Structure & Synthesis (I) PDF

RNA Structure & Synthesis (I) Learning objectives: 1. Describe the structure of RNA 2. Explain the properties of prokaryotic RNA polymerase 3. Explain the steps in RNA synthesis 4. Explain the transcription from bacterial operons There are 3 types of RNA needed for protein synthesis 1. Ribosoma...

RNA Structure & Synthesis (I) Learning objectives: 1. Describe the structure of RNA 2. Explain the properties of prokaryotic RNA polymerase 3. Explain the steps in RNA synthesis 4. Explain the transcription from bacterial operons There are 3 types of RNA needed for protein synthesis 1. Ribosomal RNA (rRNA); 2. Transfer RNA (tRNA): 3. Messenger RNA (mRNA): Structure of RNA 1. Ribosomal RNA (rRNAs)
rRNAs are found in association with several proteins as components of the ribosomes – the complex structures that serve as the sites for protein synthesis.
Prokaryotic rRNA: 23S, 16S and 5S
Eukaryotic rRNAs: 28S, 18S, 5.8S and 5S
S = Svedberg unit, related to molecular weight & shape of the compound
rRNA make up 80% of the total RNA in the cell. Prokaryotic and eukaryotic rRNAs  2. Transfer RNA (tRNA)
Smallest RNA molecules (4S)
There is at least one specific type of tRNA molecule for each of the 20 amino acids commonly found in proteins.
tRNAs make up ~15% of total RNA in the cell.
Serves as an ‘adaptor’ molecule that carries its specific amino acid – covalently attached to its 3’-end to the site of protein synthesis.
There it recognizes the genetic code word on an mRNA, which specifies the addition of its amino acid to the growing peptide chain Characteristic tRNA structure  3. Messenger RNA (mRNA)
mRNA comprises only ~5% of RNA in cell.
The most heterogeneous type of RNA in size (500 to 6000 nucleotides) and base sequence.
mRNA carries genetic information from the nuclear DNA to the cytosol
Polycistronic:
If the mRNA carries information from more than one gene
Polycistronic mRNA is characteristic of prokaryotes.
Monocistronic
the mRNA carries information from just one gene
characteristic of eukaryotes.  Eukaryotic mRNA
Special structural characteristics of eukaryotic mRNA o Long sequence of adenine nucleotides (a poly-A tail) on the 3’-end of the RNA chain o “cap” on the 5’-end consisting of a molecule of 7- methyguanosine  Structure of eukaryotic mRNA Functions: helps the small ribosomal subunit find the start codon protects the 5' end of the mRNA from degradation by exonucleases Function: delays degradation of the coding sequence of the mRNA by exonucleases Transcription of Prokaryotic Genes A. Properties of prokaryotic RNA polymerase o In bacteria, one species of RNA polymerase synthesizes all of the RNA except for the short RNA primers needed for DNA replication (RNA primer by primase) o RNA polymerase is a multisubunit enzyme that
recognizes a nucleotide sequence (the promoter region) at the beginning of a length of DNA that is to be transcribed.
Makes complementary RNA copy of the DNA template strand
Recognizes the end of the DNA sequence to be transcribed (terminal region)  RNA is synthesized from its 5’→3’ end  Transcription by RNA polymerase involves a core enzyme and several auxiliary proteins; 1. Core enzyme 2. Holoenzyme 3. Termination factor 1. Core enzyme 2α Required for enzyme assembly β' Template binding β 5’3’ RNA polymerase activity
It cannot recognize the promoter region on the DNA template
The in vivo function of a fifth subunit, Ω, is unclear 2. Holoenzyme
The σ subunit (“sigma factor”) enables RNA polymerase to recognize promoter regions on the DNA The σ subunit + Core enzyme = holoenzyme
Different σ factors recognize different groups of genes 3. Termination factor
Some regions on the DNA that signal the termination of transcription are recognized by the RNA polymerase itself
Other are recognized by specific termination factors >> rho (ρ) factor of E.coli  Steps in RNA synthesis (Prokaryotes)
Three phases: Initiation, elongation and termination 1. Initiation
Binding of RNA polymerase holoenzyme to Promoter region.
The prokaryotic promoter contains characteristic consensus sequences.
Those that are recognized by prokaryotic RNA polymerase σ factors include: a. -35 sequence b. Pribnow box  a. -35 sequence
A consensus sequence (5’-TTGACA-3’) is centered about 35 bases to the left of the transcription start site
is the initial point of contact for the holoenzyme, and a closed complex is formed  b. Pribnow box
The holoenzyme moves and covers a second consensus sequence (5'-TATAAT-3'), centered at about –10
is the site of initial DNA melting (unwinding). 
A mutation in either the –10 or the –35 sequence can affect the transcription of the gene controlled by the mutant promoter. 2. Elongation
Holoenzyme recognized promoter region
RNA polymerase begins to synthesize a transcript of DNA sequence (usually beginning with a purine)
The elongation phase is said to begin when the transcript exceeds ten nucleotides in length.
σ subunit is released. Elongation (cont’d…)
RNA polymerase
does not require a primer
Does not appear to have proofreading activity
RNA polymerase uses ribonucleoside triphosphates and releases pyrophosphate each time a nucleotide is added to the growing chain 
Binding of RNA polymerase to the DNA template results in a local unwinding of the DNA helix.
This process can generate supercoils that can be relaxed by DNA topoisomerases I and II 3. Termination
The process of elongation of the RNA chain continues until a termination signal is reached.
ρ-dependent termination: http://highered.mheducation.com/sites/dl/free/0072835 125/126997/animation21.html An additional protein, Rho (ρ) factor is required for the release of the RNA product
ρ-independent termination: 
ρ-independent termination: Action of antibiotics
Some antibiotics prevent bacterial cell growth by inhibiting RNA synthesis
Example: Rifampin
Inhibits transcription initiation by binding to the β- subunit of prokaryotic RNA polymerase
Interfering with the formation of the 1st phosphodiester bond Transcription from bacterial operons
In bacteria, the structural genes that codes for enzymes of metabolic pathway are often found grouped together on the chromosome together with the regulatory genes that determine their transcription as a single long piece of mRNA.
The genes are, thus, coordinately expressed.
This entire package is referred as operon.
Example: lactose operon of E.coli The lactose (lac) operon
lac operon codes for 3 enzymes involved in the catabolism of the sugar lactose:
lacZ gene
codes for β-galactosidase
Hydrolyzes lactose to glucose and galactose
lacY gene
codes for a permease
Facilitate the movement of galactose into the cell
lacA gene
code for thiogalactoside transacetylase
Physiologic function is unknown
All these enzymes are produced when lactose is available 
The regulatory portion of the operon consists of
the catabolite gene activator protein (CAP, sometimes called the cAMP regulatory protein or CRP) binding site
Promoter (P) region where RNA polymerase binds
Operator (O) site Glucose available ; No lactose
lacI gene codes for repressor protein
Repressor protein binds to operator site
Interferes RNA polymerase progresses and block transcription from the structural genes (negative regulation) When only lactose is available
Small amount of lactose in converted to allolactose.
Allolactose binds to repressor protein, leads to conformation change  repressor cant bind to operator
No glucoseadenynyl cyclase is activecAMP bind to CAP protein
cAMP-CAP complex binds to the CAP binding site, allows RNA polymerase to initiate transcription (positive regulation) When both glucose and lactose are present
Adenynyl cyclase is deactivated in the presence of glucose,
No cAMP-CAP complex can form, CAP binding site remain empty.
RNA polymerase is, therefore, unable to effectively initiate transcription, even though the repressor is not bound to the operator region.
Therefore, the 3 genes are not expressed