Medical Biology: Transcription and Translation

• Medical Biology and Genetics focuses on transcription and translation.
• The 5th week is the focus.

Gene Expression

• Converting the information in DNA molecules to protein molecules is gene expression.
• Genes convert to mRNA then to protein.
• Transcription is the process of transferring information carried by DNA to RNA molecules.
• Translation is translating the nucleotide sequence of mRNA to the amino acid sequence of a protein.

Central Dogma

• Genetic information in DNA is transferred to RNA then protein.
• Messenger RNA (mRNA) carries information from DNA to ribosomes.
• Transfer RNA (tRNA) translates mRNA sequences into amino acids.
• Ribosomal RNA (rRNA) has catalytic and structural roles.
• Replication is the process of copying DNA.
• Transcription is synthesizing mRNA from DNA.
• Translation is synthesizing protein from mRNA, occurring in ribosomes.

RNA Synthesis - Transcription

• Transcription is RNA synthesis from a DNA template, producing messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).
• RNA has ribose instead of deoxyribose.
• RNA contains uracil instead of thymine.
• RNA's secondary structure is created by folding.
• RNA is single-stranded, except in some RNA viruses.

Nucleic Acids

• DNA contains deoxyribose which consists of A, T, G, and C.
• RNA contains ribose which consists of A, U, G, and C.

Types of RNA

• Coding RNA molecules include mRNA.
• Noncoding RNA molecules include tRNA and rRNA.
• Small RNAs have roles in mRNA splicing, rRNA processing, and gene expression regulation.

mRNA

• mRNA carries genetic information from DNA to ribosomes for protein synthesis.

tRNA

• tRNA transfers amino acids to the ribosome to be assembled into a protein.

rRNA

• rRNA is a component of ribosomes, with eukaryotic ribosomes being 80S.

Additional Facts About RNA

• Unfolded messenger RNAs are found in bacteria and archaea, lasting briefly until ribonucleases degrade them.
• Stable RNAs like rRNAs and tRNAs are long-lived due to their secondary structures preventing ribonuclease attacks.
• The rapid turnover of mRNAs in bacteria and archaea allows adaptation to environmental changes.
• It also halts the translation of unneeded mRNA products.

Stages of RNA Synthesis

• RNA polymerase binds to promoter sequences on the DNA template.
• RNA synthesis is initiated.
• The polynucleotide chain elongates.
• Synthesis terminates.

RNA Polymerase and Transcription

• RNA Polymerase facilitates transcription.
• RNA Polymerase catalyzes polymerization of Ribonucleoside triphosphates (NTPs) creating phosphodiester bonds.
• RNA Polymerase synthesizes RNA chains in the 5' to 3' direction.
• RNA chain and template DNA strands are antiparallel to each other.
• Prokaryotic organisms use a single RNA polymerase.

RNA Polymerase Composition

• Prokaryotic RNA Polymerase is an enzyme complex of six polypeptides.
• Bacterial RNA Polymerase has 2α, β, β', w, and σ subunits.
• The core RNA polymerase is composed of 2 a, 1 β, 1 β', and 1 w (without sigma).
• The sigma subunit recognizes special promoter sequences on DNA.
• When newly synthesized RNA reaches a certain length, the sigma (σ) subunit separates from the other subunits.

Transcription - Bacteria

• Transcription is catalyzed by RNA polymerase
• Polymerization is powered by the hydrolysis of the two energy-rich phosphate bonds of incoming ribonucleoside triphosphates.
• No priming is needed.
• Ribonucleoside triphosphates attach to the 3'-OH on the ribose of the preceding nucleotide.
• Chain growth occurs in the 5' to 3' just as in DNA synthesis.
• The newly synthesized RNA strand runs antiparallel to the DNA template.

Bacterial Transcription

• Transcription continues until reaching specific sequences called transcription terminators.
• Transcription usually occurs on small DNA units, often single genes, unlike DNA replication.
• Different genes are transcribed at various rates as needed by the cell.
• Transcription is highly regulated.

RNA Polymerases and Promoters

• Subunits create the RNA polymerase holoenzyme complex.
• The sigma factor dissociates from the RNA polymerase core and only serves to recognize the DNA site for transcription.
• RNA polymerase must recognize initiation sites called promoters to transcribe.
• Promoters are recognized by sigma (σ) in bacteria.
• When the RNA polymerase binds to the promoter DNA helix, the helix opens and the polymerase moves.

Sigma Factors, Consensus Sequences, and Transcriptional Termination

• E. coli - promoter sequences are recognized by the same sigma factor called σ70.
• Sigma recognizes two highly conserved promoter regions upstream of the transcription start site.
• The -10 region, 10 bases upstream, also known as the Pribnow box - TATAAT box consensus sequence.
• The -35 consensus sequence TTGACA, 35 bases upstream of the start site.
• Promoters with -10 and -35 consensus sequences are known as strong promoters.

Transcription and Polycistronic mRNA

• Transcriptional units can be RNA transcribed from a single gene or from multiple cotranscribed genes.
• Genes include both protein-encoding and non-translated RNAs (ribosomal or transfer RNAs).
• Prokaryotic cells generate three rRNA size classes: 16S, 23S, and 5S; their genes are cotranscribed with a tRNA gene.
• Polycistronic mRNA
  • Prokaryotic cells often have genes encoding several enzymes from a particular metabolic pathway.
  • Genes for the same biochemical pathway or related functions are assembled into an operon allowing coordinated expression.
  • During transcription, RNA polymerase transcribes the entire gene set into a polycistronic mRNA.
  • Multiple open reading frames contain portions of the mRNA that can actually encode amino acids.
  • The polypeptides are synthesized sequentially by the same ribosome.

Termination of Transcription

• Bacteria - termination signal = GC-rich sequence with an inverted repeat and a central nonrepeating segment.
• After a DNA sequence is transcribed, the RNA produces a stem-loop structure by intra-strand base pairing.
• Stem-loops, followed by run adenines in the template, are strong transcription terminators.
• RNA polymerase pauses at the stem-loop, causing DNA and RNA to dissociate.
• Termination mechanism for transcription
  • Terminator protein Rho.
  • Rho doesn't bind to either RNA polymerase or DNA.
  • But Rho binds to RNA and moves down the chain toward the RNA polymerase-DNA complex
• RNA polymerase stalls at a Rho-dependent termination site - Rho causes both the RNA and RNA polymerase to be released, ending transcription.

Eukaryotic Transcription

• Archaeal and Eukaryotic RNA polymerases are similar and more complex than those of Bacteria.

• Archaea has one RNA polymerase.

  • Eukaryotes have three.

• Archeal polymerase resembles eukaryotic RNA polymerase II - includes 11-13 subunits based on species.

• Archaeal and eukaryotic promoters have important recognition sequences.

• Most important is the 6-8 base-pair "TATA" box, 18-27 nucleotides upstream from the transcriptional start site.

• The TATA box is recognized by the TATA-binding protein (TBP).

  • Archaeal recognition also has B recognition element (BRE) which are followed by archaeal Transcription factor B(TFB).

• There should be a specific initiator element at the start of transcription.

• After the TBP binds to the TATA box - TFB binds to the BRE to have RNA polymerase initiate transcription.

• This Process is similar in eukaryotes however they need additional transcription factors for transcription.

• Transcription termination in Archaea and Eukaryotes

• Some Archaeal genes have inverted repeats with an AT-rich sequence. One includes repeats of thymines.

• Termination in eukaryotes depends on RNA polymerase with the need for a termination factor protein.

RNA Processing - Eukaryotes & Archaea

• Eukaryotes have many genes.
  • Two coding and non coding regions. Coding sequences are exons and noncoding are introns.
  • RNA must process in mature RNAs so that translation can take place.
  • Primary transcript occurs when introns are removed.
  • Mature mRNA occurs when only exons are present within.

RNA Processing - Splicing

• Introns are removed in spliced. Exons are joined in this process.
• Process happens in the nucleus
  • The process has to have both RNA and protein by the spliceosome.
• Primary transcripts in spliceosomes remove introns.
  • Mature mRNA has contiguous protein by linking flanking exons to one another.

Further RNA Processing

• Intervening sequences are rare but archaeal includes tRNA and has introns.
  • archaeal introns need to be removed after transcription to generate mature tRNA or rRNA.
• Their processing is catalzyed with a special ribosome but not the spliceosome.
• In eukarya, two processing steps take place within the nucleus before splicing
  • Capping is complete once transcription is done.
    • Capping creates a methylated guanine nucleotide at the 5'-phosphate end.
• Orientation is then converted reverse relative to to the rest.
• A poladenylation
  • trimming has a 3' end
  • poly(A) stabilizes mRNA from nuclease attack
    • mRNA have to be removed bfore degradation.