Chapter Eleven Transcription of the Genetic Code: The Biosynthesis of RNA

Transcription Overview of Transcription • synthesized on a DNA template, catalyzed by DNA-dependent RNA polymerase • ATP, GTP, CTP, and UTP are required, as is Mg2+ • no RNA primer is required • the DNA base sequence contains signals for initiation and termination of RNA synthesis; the enzyme binds to and moves along the DNA template in the 3’ -> 5’ direction the RNA chain is synthesized in the 5’ -> 3’ direction • the DNA template is unchanged

Transcription in Prokaryotes E. coli RNA Polymerase: molecular weight about 500,000 four different types of subunits: ,  , ’, and s the core enzyme is 2’ the holoenzyme is 2’s the role of the s subunit is recognition of the promoter locus; the s subunit is released after transcription begins of the two DNA strands, the one that serves as the template for RNA synthesis is called the template strand or antisense strand; the other is called the coding (or nontemplate) strand or sense strand the holoenzyme binds to and transcribes only the template strand

The Basics of Transcription

Promoter Sequence Simplest of organisms contain a lot of DNA that is not transcribed RNA polymerase needs to know which strand is template strand, which part to transcribe, and where first nucleotide of gene to be transcribed is Promoters-DNA sequence that provide direction for RNA polymerase

Promoter Sequence

How does RNA polymerase know where to begin transcription? Polymerase moves along template strand from 3’-5’ and RNA is formed from 5’-3’ Binding site for polymerase is upstream of start of transcription – away from 5’ of coding strand Promoter sequence is based on coding strand and RNA polymerase is binding to template strand

How does RNA polymerase know where to begin transcription? Promoter are upstream towards 5’ of coding and 3’ template strand The first base to be incorporated is at position + 1 = transcription start site All nucleotides upstream from this start site are given –ve numbers The first promoter element is about 10 bases upstream is – 10 region or pribnow box

How does RNA polymerase know where to begin transcription? After PB 16-18 bases are variable Next promoter element is about 35 bases upstream of TSS = -35 region or -35 element Area from -35 element to TSS = core promoter Upstream of core element is UP element = enhances binding of RNA polymerase Region from UP element to TSS = extended promoter BASE SEQUENCE of promoter is A and T

Chain Initiation First phase of transcription is initiation Initiation begins when RNA polymerase binds to promoter and forms closed complex After this, DNA unwinds at promoter to form open complex, which is required for chain initiation

Initiation and Elongation Transcription

Chain Elongation (Cont’d)

Chain Elongation After strands separated, transcription bubble of ~17 bp moves down the DNA sequence to be transcribed RNA polymerase catalyzes formation of phosphodiester bonds between the incorp. ribonucleotides Topoisomerases relax supercoils in front of and behind transcription bubble

Chain Termination Two types of termination mechanisms: • intrinsic termination- controlled by specific sequences, termination sites Termination sites characterized by two inverted repeats The inverted repeats are sequences of bases that are complementary such that they can loop back on themselves. Dna codes a series of uracils. The A-U base pairs between template strand forming hairpin loop. These are weakly bonded compared with gc pairs and rna dissociates from transcription bubble ending transcription

Chain Termination Other type of termination involves rho () protein Rho-dependent termination sequences cause hairpin loop to form Rho sequences form hair loop and rho protein will stall polymerase from proceeding

Transcription Regulation in Prokaryotes In prokaryotes, transcription regulated by: • alternative s factors enhancers operons transcription attenuation

Alternative s factors Viruses and bacteria exert control over which genes are expressed by producing different s-subunits that direct the RNA polymerase to different genes.

Enhancers Certain genes include sequences upstream of extended promoter region These genes for ribosomal production have 3 upstream sites, Fis sites Class of DNA sequences that do this are called enhancers Bound by proteins called transcription factors

Elements of a Bacterial Promoter

Operon Operon: a group of operator, promoter, and structural genes that codes for proteins the control sites, promoter, and operator genes are physically adjacent to the structural gene in the DNA the regulatory gene can be quite far from the operon operons are usually not transcribed all the time

Example of Operon system b-Galactosidase, an inducible protein coded for by a structural gene, lacZ structural gene lacY codes for lactose permease structural gene lacA codes for transacetylase expression of these three structural genes is controlled by the regulatory gene lacI that codes for a repressor

Transcription in Eukaryotes is complex Three RNA polymerases are known - each transcribes a different set of genes and recognizes a different set of promoters: • RNA Polymerase I- found in the nucleolus and synthesizes precursors of most rRNAs • RNA Polymerase II- found in the nucleoplasm and synthesizes mRNA precursors • RNA Polymerase III- found in the nucleoplasm and synthesizes tRNAs, other RNA molecules involved in mRNA processing and protein transport Prokaryotes have single RNA polymerase responsible for three kinds of prokayotic RNA - m RNA, t RNA and r RNA. THE polymerase switch sigma factors to interact with different promoters but core polymerase stays same. All three eukaryotic RNA are large and consist of 10 or more subunits. Their overall structure differs but have two larger subunits that share homology with ß and ß1 of prokaryotic RNA polymerase. There are no sigma subunits to direct polymerases to promoters.

RNA Polymerase II Most studied in the polymerases Consists of 12 subunits RPB- RNA Polymerase B RPB1-RPB12. I, II, III also known as A, B and C

How does Pol II Recognize the Correct DNA? Four elements of the Pol II promoter .

Pol II promoters Variety of upstream elements – activators and silencers GC box (-40) – Consensus sequence – GGGCGG CAAT box (extending to – 110) – Consensus sequence - GGCCAATCT

Pol II promoters Second element found at -25 = TATA box has consensus sequence of TATAA (T/A) Transcription start site at position + 1 surrounded by a sequence called initiator element (Inr) Inititator and TATA box = core promoter Fourth element – downstream regulator - rare ALL four are not present in all eukaryotes. Core promoter is most consistent in most species and genes

Initiation of Transcription Transcription factor -Any protein regulator of transcription that is not a subunit of Pol II Initiation begins by forming the preinitiation complex - Transcription control is based here General transcription factors

Transcription Order of Events The phosphorylated Pol II synthesizes RNA Leaves the promoter region behind GTFs are left at the promoter or dissociate from Pol II Less is known about eukaryotes than prokaryotes. Pol II gets dephosphorylated and recycles back

Elongation and Termination Elongation is controlled by: pause sites - where RNA Pol will hesitate positive transcription elongation factor (P-TEF) and negative transcription elongation factor (N-TEF) Termination begins by stopping RNA Pol; the eukaryotic consensus sequence for termination is AAUAAA Elongation factors increase the productive form and decrease abortive form

Gene Regulation Enhancers and silencers- regulatory sequences that augment or diminish transcription, respectively DNA looping brings enhancers into contact with transcription factors and polymerase

Response elements Response elements are enhancers that respond to certain metabolic factors • heat shock element (HSE) • glucocorticoid response element (GRE) • metal response element (MRE) • cyclic-AMP response element (CRE)

Structural Motifs in DNA-Binding Proteins Most proteins that activate or inhibit RNA Pol II have two functional domains: DNA-binding domain transcription-activation domain DNA-Binding domains have domains that are either: • Helix-Turn-Helix (HTH) • Zinc fingers • Basic-region leucine zipper Interact through hydrogen bonding, electrostatic attractions and hydrophobic interactions

Helix-Turn-Helix Motif Hydrogen bonding between amino acids and DNA

Zinc Finger Motif Motif contains 2 cysteines and 2 His – after every 12 amino acids Zinc binds to the repeats

Basic Region Leucine Zipper Motif Many transcription factors contain this motif - CREB Half of the protein composed of basic region of conserved Lys, Arg, and His Half contains series of Leu

Post Transcriptional RNA Modification tRNA, rRNA, and mRNA are all modified after transcription to give the functional form the initial size of the RNA transcript is greater than the final size because of the leader sequences at the 5’ end and the trailer sequences at the 3’ end Modifications trimming of leader and trailer sequences addition of terminal sequences (after transcription) modification of specific bases (particularly in tRNA) the types of processing in prokaryotes can differ greatly from that in eukaryotes, especially for mRNA

Modification of tRNA Transfer RNA- trimming, addition of terminal sequences, and base modification - take place methylation and substitution of sulfur for oxygen are the two most usual types of base modification

Modification of rRNA Ribosomal RNA processing of rRNA - methylation and trimming to the proper size

Modification of mRNA Capping of the 5’ end with an N-methylated guanine A polyadenylate “tail” that is usually100-200 nucleotides long, is added to the 3’ end before the mRNA leaves the nucleus This tail protects the mRNA from nucleases and phosphatases

Organization of Split Genes in Eukaryotes

Modification of mRNA Eukaryote genes frequently contain intervening base sequences that do not appear in the final mRNA of that gene product Expressed DNA sequences are called exons Intervening DNA sequences that are not expressed are called introns These genes are often referred to as split genes

The Splicing Reaction Exons are separated by intervening intron When the exons are spliced together - lariat forms in the intron

Ribozymes The first ribozymes discovered included those that catalyze their own self-splicing ribozymes have been discovered that are involved in protein synthesis Group I and II

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