Transcription of the Genetic Code: The Biosynthesis of RNA Mar 1, 2015 CHEM 281

Transcription

Transcription in Prokaryotes  E. coli RNA Polymerase:  molecular weight about 500,000  four different types of subunits: , ,  ’, and   the core enzyme is  2  ’  the holoenzyme is  2  ’   the role of the  subunit is recognition of the promoter locus; the  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

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

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  Movement and dissociation requires ATP

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.

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   -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

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 the 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

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

Non-Coding RNAs  As much as 98% of transcriptional output from human genomes may be comprised of non-coding RNAs (ncRNA)  Linked to: regular transcription, gene silencing, replication, processing of RNA, RNA modification, translation, protein stabilization, protein translocation  Two main types: Micro RNA (miRNA), and Small Interfering RNA (siRNA)

SiRNAs are formed in a way similar miRNA

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)

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  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

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

Modification of tRNA  Transfer RNA- the precursor of several tRNAs 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

Posttranscriptional Modification of tRNA Precursor

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