Transcription in Prokaryotic (Bacteria) The conversion of DNA into an RNA transcript requires an enzyme known as RNA polymerase RNA polymerase – Catalyzes the formation of a phosphodiester bond between the 3’ end of the growing mRNA chain and the new ribonucleotides – Transcription must occur in the 5’ to 3’ direction – No primer is needed to begin transcription

Transcription in Bacteria The template strand on DNA is what is “read” by RNA polymerase into a message. The non-template strand is confusingly referred to also as the coding strand because the DNA sequence in this strand is complementary to the message which is made into Protein.

RNA Polymerase Structure X-ray crystallography indicates that – Large globular enzyme – Several channels in the interior Core enzyme – Just RNA polymerase – Active site for catalysis Holoenzyme – Sigma plus RNA polymerase Holoenzyme

Initiation of Transcription in Bacteria Promoters – Sequences of DNA which “inform” RNA polymerase where to begin transcription – Located on non-template strand – 40 to 50 base pairs in length – All have a TATAAT sequence -10 box centered 10 bases away from the site where transcription begins (+1) – All have a TTGACA -35 box located 35 bases away from the +1 site – Sequences inside promoter but outside -10 or -35 vary widely among genes

Terms Downstream – DNA located in the direction in which RNA polymerase will transcribe Upstream – DNA located in the opposite direction in which RNA polymerase will transcribe TTGACTATAAT Promoter Upstream Downstream Gene

Initiation of Transcription in Bacteria Sigma makes the initial contact with DNA that starts transcription in bacteria Once sigma has bound to the DNA the helix opens and the template is threaded through a channel NTPs enter a different channel and diffuse to the active site Incoming NTPs base pair complementary with the template strand – A base pairs with U not T

Opens the helix Steers the template and non-template strands into the correct channels

Termination of Transcription in Bacteria DNA sequence functions as a transcription termination signal Makes a stretch of RNA which as soon as it is synthesized can base pair with itself – Hairpin loop – RNA secondary structure The formation of the hairpin disrupts the interaction between RNA polymerase and the RNA transcript resulting in the physical separation

Termination of Transcription in Bacteria

Transcription in Eukaryotic Cells Transcription of eukaryotic cells is more complex and requires more proteins and regulators Transcription factors The initiation of transcription is accomplished by basal transcription factors – Function analogous to sigma – Can interact with DNA independently of RNA polymerase

Transcription Regulation What we know from prokaryotes: – Several related genes can be transcribed together (ie. lac operon) – Need RNA Polymerase to recognize a promoter region Why eukaryotes are different: – Genes are nearly always transcribed individually – 3 RNA Polymerases occur, requiring multiple proteins to initiate transcription

Transcription Regulation Typical prokaryotic promoter: recognition sequence + TATA box -> RNA Polymerase attachment -> transcription Typical eukaryotic promoter: recognition sequence + TATA box + transcription factors -> RNA Polymerase II attachment -> transcription

Transcription Regulation RNA polymerase interacts w/promoter, regulator sequences, & enhancer sequences to begin transcription – Regulator proteins bind to regulator sequences to activate transcription Found prior to promoter – Enhancer sequences bind activator proteins Typically far from the gene Silencer sequences stop transcription if they bind with repressor proteins

Activator: A transcriptional activator is a protein that increases gene transcription of a gene or set of genes. Most activators are DNA-binding proteins. Most activators function by binding sequence-specifically to a DNA site located in or near a promoter and making protein-protein interactions with the general transcription machinery (RNA polymerase and general transcription factors). Transcription factor : is a protein that binds to specific DNA sequences, thereby controlling the flow (or transcription) of genetic information from DNA to mRNA. Transcription factors perform this function alone or with other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase (the enzyme that performs the transcription of genetic information from DNA to RNA) to specific genes

Repressor: repressor is a DNA-binding protein that regulates the expression of one or more genes by binding to the operator and blocking the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes. This blocking of expression is called repression. Repressor proteins are coded for by regulator genes. Repressor proteins then attach to a DNA segment known as the operator. By binding to the operator, the repressor protein prevents the RNA polymerase from creating messenger RNA

Transcription in Eukaryotic Cells Eukaryotic genes contain promoters Promoters are much more diverse and complex Many promoters recognized by RNA pol II include a TATA box at -30 Some promoters recognized by RNA pol II do not contain a TATA box RNA pol I and RNA pol III interact with entirely different promoters

Transcription in Eukaryotic Cells Unlike prokaryotic cells eukaryotic cells must process the mRNA in the nucleus before it can be made into a protein Three major modifications are made – Intron splicing – Addition of poly A tail – Additon of 5’ cap

Exons & Introns Exons – Coding regions of DNA – Expressed as proteins Introns – Non-coding regions of DNA – Intervening

Intron Removal Introns are removed in a process known as intron splicing Splicing occurs while transcription is still underway Removed by ribozymes known as small nuclear ribonucleoproteins – snRNPs : are RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs. The action of snRNPs is essential to the removal of introns from pre-mRNA, a critical aspect of post- transcriptional modification of RNA, occurring only in the nucleus of eukaryotic cells.

1- Pre-mRNA splicing. Small nuclear RNAs (snRNAs) form a complex called a spliceosome with small nuclear ribonucleoproteins (snRNPs) and other proteins. 2- The snRNAs bind to specific nucleotides in the introns of a pre-mRNA. 3- The RNA transcript is cut, releasing the introns and splicing the exons together, producing mature mRNA.

Adding a Cap and a Tail 5’ cap The process of 5′ capping is vital to creating mature messenger RNA, which is then able to undergo translation. Capping ensures the messenger RNA's stability while it undergoes translation in the process of protein synthesis, and is a highly regulated process that occurs in the cell nucleus. Because this only occurs in the nucleus, mitochondrial and chloroplast mRNA are not capped. Poly A tail – Polyadenylation is the addition of a poly(A) tail to an RNA molecule. The poly(A) tail consists of multiple adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation. It, therefore, forms part of the larger process of gene expression.

AspectBacteriaEukaryotes RNA polymeraseOne Three; each produces a different class of RNA Promoter Structure -35; -10 Complex and variable; often has -30 Proteins which contact promoter Sigma; many types Many basal transcription factors RNA processingNone Extensive 1. Intron splicing 2. Cap and poly A tail Comparing Transcription: Bacteria vs. Eukaryotes