Paul D. Adams University of Arkansas Mary K. Campbell Shawn O. Farrell 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 Mg 2+ no RNA primer is required the RNA chain is synthesized in the 5’ -> 3’ direction; the nucleotide at the 5’ end of the chain retains its triphosphate (ppp) group 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 DNA template is unchanged

Transcription in Prokaryotes E. coli RNA Polymerase: molecular weight about 500,000 four different types of subunits: , ,  ’, and  core enzymethe core enzyme is  2  ’ holoenzymethe holoenzyme is  2  ’  promoterlocusthe role of the  subunit is recognition of the promoter locus; the  subunit is released after transcription begins antisense strand sense strandof 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

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 in Transcription

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 Elongation (Cont’d)

Chain Termination Two types of termination mechanisms: intrinsic termination- controlled by specific sequences, termination sites Termination sites characterized by two inverted repeats

Chain Termination (Cont’d) Other type of termination involves rho (  ) protein Rho-dependent termination sequences cause hairpin loop to form

Transcription Regulation in Prokaryotes In prokaryotes, transcription regulated by: alternative  factors enhancers operons transcription attenuation Alternative  factors Viruses and bacteria exert control over which genes are expressed by producing different  -subunits that direct the RNA polymerase to different genes.

Control by Different  Subunits

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: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  -Galactosidase,  -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

How Does Repression Work Repressor protein made by lacI gene forms tetramer when it is translated Repressor protein then binds to operator portion of operon Operator and promoter together are the control sites

Binding Sites On the lac operon Lac operon is induced when E. coli has lactose as the carbon source Lac protein synthesis repressed by glucose (catabolite repression) E. coli recognizes presence of glucose by promoter as it has 2 regions: RNA polymerase binding site, catabolite activator protein (CAP) binding site

Binding Sites On lac operon (Cont’d)

Catabolite Repression CAP forms complex with cAMP Complex binds at CAP site RNA polymerase binds at available binding site, and transcription occurs

Basic Control Mechanisms in Gene Control Control may be inducible or repressive, and these may be negatively or positively controlled

Control of the trp operon Trp operon codes for a leader sequence (trpL) and five polypeptides The five proteins make up 4 different enzymes that catalyze the multistep process that converts chorisimate to tryptophan

Alternative 2˚ structures Can Form in trp Operon These structures can form in the leader sequence Pause structure- binding between regions 1 and 2 Terminator loop- binding between regions 3 and 4 Antiterminator structure- Alternative binding between regions 2 and 3

Attenuation in the trp operon Pause structure forms when ribosome passes over Trp codons when Trp levels are high Ribosome stalls at the Trp codon when trp levels are low and antiterminator loop forms

Transcription in Eukaryotes 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

RNA Polymerase II Most studied on the polymerases Consists of 12 subunits RPB- RNA Polymerase B

How does Pol II Recognize the Correct DNA? Four elements of the Pol II promoter allow for this phenomenon

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

General Transcription Initiation Factors

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

Elongation and Termination Elongation is controlled by: pause sites, where RNA Pol will hesitate anti-termination, which proceeds past the normal termination point 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

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

Eukaryotic Gene Regulation 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) Response elements all bind proteins (transcription factors) that are produced under certain cell conditions

Response Elements

Activation of transcription Via CREB and CBP Unphosphorylated CREB does not bind to CREB binding protein, and no transcription occurs Phosphorylation of CREB causes binding of CREB to CBP Complex with basal complex (RNA polymerase and GTFs) activates transcription

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

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

Zinc Finger Motif Motif contains 2 cysteines and 2 His 12 amino acids later Zinc binds to the repeats

Basic Region Leucine Zipper Motif Many transcription factors contain this motif, such as CREB (Biochemical Connections, page 315) Half of the protein composed of basic region of conserved Lys, Arg, and His Half contains series of Leu Leu line up on one side, forming hydrophobic pocket

Helical Wheel Structure of Leucine Zipper

Transcription Activation Domains acidic domains- rich in Asp and Glu. Gal4 has domain of 49 amino acids, 11 are acidic glutamine-rich domains- Seen in several transcription factors. Sp1 has 2 glutamine-rich domains, one with 39 Glu in 143 amino acids proline-rich domains- Seen in CTF-1 (an activator). It has 84 amino acid domain, of which 19 are Pro

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 the types of processing in prokaryotes can differ greatly from that in eukaryotes, especially for mRNA Modifications trimming of leader and trailer sequences addition of terminal sequences (after transcription) modification of the structure of specific bases (particularly in tRNA)

Posttranscriptional Modification of tRNA Precursor

Modification of tRNA Transfer RNA- the precursor of several tRNAs is can be transcribed as one long polynucleotide sequence trimming, addition of terminal sequences, and base modification all 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 is primarily a matter of methylation and trimming to the proper size in prokaryotes, 3 rRNAs in one intact ribosome in Eukaryotes, ribosomes have 80s, 60s, and 40s subunits base modification in both prokaryotes and eukaryotes is primarily by methylation

Modification of mRNA Includes the capping of the 5’ end with an N-methylated guanine that is bonded to the next residue by a 5’ -> 5’ triphosphate. Also, 2’-O-methylation of terminal ribose(s)

mRNA Modification A polyadenylate “tail” that is usually nucleotides long, is added to the 3’ end before the mRNA leaves the nucleus This tail protects the mRNA from nucleases and phosphatases Eukaryote genes frequently contain intervening base sequences that do not appear in the final mRNA of that gene product exonsExpressed DNA sequences are called exons intronsIntervening DNA sequences that are not expressed are called introns These genes are often referred to as split genesThese genes are often referred to as split genes

Organization of Split Genes in Eukaryotes

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

Ribozymes The first ribozymes discovered included those that catalyze their own self-splicing More recently, ribozymes have been discovered that are involved in protein synthesis Group I ribozymes require an external guanosine example: pre-rRNA of the protozoan Tetrahymena (next screen) Group II ribozymes display a lariat mechanism similar to mRNA splicing no requirement for an external nucleotide

Self-splicing of pre-rRNA