Transcription: DNA-Directed RNA Synthesis

• Transcription is the DNA-directed synthesis of RNA and the first stage of gene expression.

Molecular Components of Transcription

• Messenger RNA (mRNA) carries information from DNA to the protein-synthesizing machinery.
• RNA polymerase separates DNA strands and joins RNA nucleotides complementary to the DNA template.
• RNA polymerase assembles a polynucleotide only in the 5' → 3' direction, adding to the 3' end.
• Unlike DNA polymerases, RNA polymerases do not need a pre-existing primer to start a chain.
• Specific nucleotide sequences on DNA mark where gene transcription begins and ends.
• The promoter is the DNA sequence where RNA polymerase attaches and initiates transcription.
• The terminator is the sequence that signals the end of transcription in bacteria.
• "Downstream" refers to the direction of transcription, and "upstream" refers to the opposite direction.
• The transcription unit is the stretch of DNA downstream from the promoter that is transcribed into an RNA molecule.
• Bacteria have a single type of RNA polymerase that synthesizes mRNA and other RNAs for gene expression, such as ribosomal RNA.
• Eukaryotes have at least three types of RNA polymerase, with RNA polymerase II used for pre-mRNA synthesis.
• Other eukaryotic RNA polymerases transcribe RNA molecules that are not translated into protein.

Synthesis of an RNA Transcript

• Three stages: initiation, elongation, and termination of the RNA chain.

RNA Polymerase Binding and Initiation of Transcription

• The promoter sequence includes the transcription start point, where RNA polymerase starts mRNA synthesis.
• The promoter extends several dozen nucleotide pairs upstream from the start point.
• RNA polymerase binds in a precise location and orientation on the promoter, through interactions with transcription factors.
• This binding determines the transcription start point and the DNA strand used as the template.
• Eukaryotic cells use transcription factors to mediate initiation by RNA polymerase II.
• A eukaryotic promoter includes a TATA box (containing TATA) about 25 nucleotides upstream from the transcriptional start point.
• Transcription factors, including one recognizing the TATA box, must bind to the DNA before RNA polymerase II can bind.
• Additional transcription factors and RNA polymerase II form the transcription initiation complex.
• RNA polymerase II then unwinds the DNA double helix, and RNA synthesis begins at the start point.

Elongation of the RNA Strand

• As RNA polymerase moves, it untwists the double helix, exposing 10-20 DNA nucleotides at a time to pair with RNA nucleotides.
• The enzyme adds nucleotides to the 3' end of the growing RNA molecule.
• The newly synthesized RNA molecule peels away from the DNA template, and the DNA double helix re-forms.
• Transcription progresses at a rate of about 40 nucleotides per second in eukaryotes.
• Several RNA polymerase molecules can simultaneously transcribe a single gene.
• This increases the amount of mRNA transcribed, which helps the cell make the encoded protein in large amounts.

Termination of Transcription

• Bacteria and eukaryotes use different methods to terminate transcription.
• In bacteria, transcription proceeds through a terminator sequence in the DNA.
• The transcribed terminator (an RNA sequence) functions as the termination signal, causing the polymerase to detach and release the transcript, without further modification.
• In eukaryotes, RNA polymerase II transcribes a DNA sequence called the polyadenylation signal sequence, which specifies a polyadenylation signal (AAUAAA) in the pre-mRNA.
• Proteins bind to the AAUAAA sequence, then cut the RNA transcript free from the polymerase 10-35 nucleotides downstream from the AAUAAA, releasing the pre-mRNA.
• RNA polymerase II continues to transcribe, while enzymes degrade the RNA made after cleavage, until they catch up to the polymerase and it dissociates.

Eukaryotic Cells Modify RNA After Transcription

• Enzymes in the eukaryotic nucleus modify pre-mRNA before it is sent to the cytoplasm.
• During RNA processing, both ends of the primary transcript are altered, and certain interior sections are cut out and the remaining parts are spliced together.
• It produces an mRNA molecule ready for translation.

Alteration of mRNA Ends

• The 5' end, synthesized first, receives a 5' cap, a modified guanine nucleotide added after the first 20-40 nucleotides have been transcribed.
• An enzyme adds 50-250 adenine nucleotides to the 3' end, forming a poly-A tail.
• The 5' cap and poly-A tail facilitate mRNA export from the nucleus and protect the mRNA from degradation by hydrolytic enzymes.
• They also help ribosomes attach to the 5' end of the mRNA in the cytoplasm.
• Eukaryotic mRNA molecules have untranslated regions (UTRs) at the 5' and 3' ends, which are not translated into protein, but have other functions like ribosome binding.

Split Genes and RNA Splicing

• A large portions of the RNA primary transcript molecules are removed and the remaining portions are reconnected which is an stage of RNA processing in the eukaryotic nucleus called RNA splicing.
• The poly-A tail and 5' cap are not translated into protein, nor are the 5' and 3' untranslated regions (UTRs).
• Introns are segments which will be swiftly described.
• The average length of a transcription unit along a human DNA molecule is about 27,000 nucleotide pairs.
• The average-sized protein of 400 amino acids requires only 1,200 nucleotides in RNA to code for it.
• Eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides
• Most noncoding sequences are interspersed between coding segments.
• The noncoding segments of nucleic acid that lie between coding regions are called intervening sequences, or introns.
• The other regions are called exons because they are eventually expressed, usually by being translated into amino acid sequences.
• Intron and exon code for both RNA sequences and the DNA.
• RNA polymerase II transcribes both introns and exons from the DNA, but the mRNA molecule that enters the cytoplasm is an abridged version.
• The introns are cut out from the molecule and the exons joined together.
• The removal of introns is accomplished by a large complex made of proteins and small RNAs called a spliceosome.
• This complex binds to several short nucleotide sequences along an intron, including key sequences at each end
• The intron is released (and rapidly degraded), and the spliceosome joins together the two exons that flanked the intron.
• The small RNAs in the spliceosome participate in spliceosome assembly and catalyze the splicing reaction.
• Like proteins, RNAs can act as catalysts.