Medical Genetics & Genomics Guri Tzivion, PhD Extension 506 BCHM 590: Fall 2015 Windsor University School of Medicine

Questions on RNA and transcription?

Transcription initiation 1. Sigma binds to specific promoter regions of DNA (-35 box and -10 box).

Eukaryotic mRNA Processing After transcription, specific regions of the primary RNA transcript are spliced out and degraded during RNA processing. Exon: Contains sequences required for protein synthesis and is NOT spliced out during RNA processing. Is part of the final mature spliced RNA transcript. Intron: Spliced out during RNA processing and degraded. Exon 1 Exon 2 Exon 3

Eukaryotic mRNA Processing Besides splicing, other steps involved in mRNA processing include: 1. Addition of a “cap” at the 5’ end of the mRNA. The “5’ cap” serves as a recognition signal for the translation machinery of the cell. 2. Addition of poly (A) tail at the 3’ end of the mRNA. Serves to stabilize the mRNA by protecting it from enzymatic degradation.

Prokaryote versus Eukaryote Transcription

BCHM 560 MDII Genetics Class 7 DNA: Structure, Replication and Regulation of Gene Expression 3. Proteins and Translation

DNA RNA protein transcriptiontranslationreplication reverse transcription Central dogma

From messenger RNA to protein: Translation The mRNA is translated by ribosomes as series of 3 letter codes designated codons. How many combinations are there?

The Code 64 codons – 20 amio acids Redundant but not ambiguous; no codon specifies more than one amino acid (but one amino acid may have >1 codon) must be read in the correct reading frame in order for the specified polypeptide to be produced

Second mRNA base First mRNA base (5 end) Third mRNA base (3 end) The Genetic Code

The Genetic Code

Evolution of the Genetic Code nearly universal: shared by the simplest bacteria, plants, fungi and animals Genes can be transcribed and translated after being transferred from one species to another

Translation Mechanism Ribosomes bind messenger RNA (mRNA) and attract transfer RNA (tRNA) to the mRNA The tRNA is covalently linked with specific amino acids (aa-tRNA complex) Complementary base pairs form between the mRNA and the aa-tRNA complex (codon- anticodon interactions) Enzymes in the ribosome catalyzes peptide bonds between the amino acids The polypeptide chain grows The polypeptide chain grows

Transfer RNA Transfer RNA molecules are short RNAs that fold into a characteristic cloverleaf pattern. Each tRNA has 3 bases that make up the anticodon. These bases pair with the 3 bases of the codon on mRNA during translation. Each tRNA has its corresponding amino acid attached to the 3’ end. Aminoacyl tRNA synthetases “charge” the tRNA with the proper amino acid.

General tRNA structure: ~ 80 nt long

Three-dimensional structure

tRNAs are activated by amino-acyl tRNA synthetases

Ribosomes tRNA molecules Exit tunnel Growing polypeptide Large subunit mRNA 3 Small subunit 5 E P A Facilitate specific coupling of anticodons with codons Made of two subunits: small and large, contain ribosomal proteins and rRNA Have binding sites for mRNA and aa- tRNA

Copyright © 2006 by Elsevier, Inc. A complex of the small ribosomal subunit and the initiator tRNA (Met) binds to the 5’end of an mRNA chain This complex moves along the mRNA molecule until it encounters a start codon (AUG) The initiation factors dissociate and the large ribosomal subunit binds the mRNA Protein Synthesis Phase 1: Initiation

Initiation is controlled differently in prokaryotic and eukaryotic ribosomes In prokaryotes a single transcript can give rise to multiple proteins Initiation

Polyribosomes (exist in prokaryotes and viruses) A single mRNA (transcript) is translated by many ribosomes simultaneously mRNA + bound ribosomes = polyribosomes (polysome) Allows fast synthesis of many copies a polypeptide

Copyright © 2006 by Elsevier, Inc. Polyribosome: multiple ribosomes can simultaneously translate a single mRNA Polyribosomes

RNA polymerase DNA Polyribosome RNA polymerase Direction of transcription mRNA 0.25  m DNA Polyribosome Polypeptide (amino end) Ribosome mRNA (5 end) Translation using polyribosomes

Messenger RNA translated on polyribosomes

Initiation in prokaryotes Requirements: The large and small ribosome subunits mRNA The initiator tRNA Three initiation factors

In prokaryotes, ribosomes bind to specific translation initiation sites. There can be several different initiation sites on a messenger RNA: a prokaryotic mRNA can code for several different proteins. Translation begins at an AUG codon, or sometimes a GUG. The modified amino acid N-formyl methionine is always the first amino acid of the new polypeptide. In eukaryotes, ribosomes bind to the 5’ cap, then move down the mRNA until they reach the first AUG and begin translation. Eukaryotic mRNAs code for only a single gene (there are a few exceptions, mainly among the eukaryotic viruses). Note that translation does not start at the first base of the mRNA. There is an untranslated region at the beginning of the mRNA, the 5’ untranslated region (5’ UTR). Initiation

Initiatiator tRNA Methionine is the first amino acids incorporated into a protein chain in both prokaryotes (modified to N- formylmethionine) and eukaryotes. Initiator tRNAs are special tRNAs recognizing the AUG (alternative start codon GUG) start codons in prokaryotes and eukaryotes. Initiator tRNAs differ from the one that inserts internal Met residues.

Met GTP Initiator tRNA mRNA 5 3 mRNA binding site Small ribosomal subunit Start codon P site 5 3 Translation initiation complex E A Large ribosomal subunit GDP Met The initiation process involves the association of the mRNA, the initiator methionine-tRNA and the small ribosomal subunit. Several additional “initiation factors” -are also involved. The large ribosomal subunit then joins the complex. Initiation in prokaryotes

Schematic model showing the binding sites on the ribosome P site (Peptidyl-tRNA binding site) E site (Exit site) mRNA binding site A site (Aminoacyl- tRNA binding site) Large subunit Small subunit EPA The assembled ribosome has one exit site and two tRNA-binding sites, designated A and P for aminoacyl and peptidyl-tRNA binding sites respectively. Only fMet-tRNA fMet can be used for initiation by the 30S subunits; all other aminoacyl-tRNAs are used for elongation by the 70S subunits.

Most of the differences in the mechanism of protein synthesis between prokaryotes and eukaryotes occur in the initiation stage, where a greater numbers of initiation factors and a scanning process are involved in eukaryotes. Most of the differences in the mechanism of protein synthesis between prokaryotes and eukaryotes occur in the initiation stage, where a greater numbers of initiation factors and a scanning process are involved in eukaryotes. The eukaryotic initiator tRNA does not become N- formylated. The eukaryotic initiator tRNA does not become N- formylated. Initiation in eukaryotes

Eukaryotic initiation factors and their identified functions: Binding to ribosomal subunitseIF6 eIF3 eIF4c Binding to the mRNAeIF4B eIF4F eIF4A eIF4E Involved in initiation tRNA delivery eIF2 eIF2B Displace other factorseIF5 In contrast to the events in prokaryotes, initiation in eukaryotes requires first association of the 40S small subunit with the initiation tRNA before the binding to the mRNA, which is facilitated by the 5’ cap. Initiation in eukaryotes

Eukaryotic mRNAs have a distinct structure at the 5’ end

Eukaryotes use a scanning mechanism to initiate translation Recognition of the AUG triggers GTP hydrolysis by eIF-2

GTP hydrolysis by eIF2 is a signal for binding of the large subunit and beginning of translation

Copyright © 2006 by Elsevier, Inc. aa-tRNA binds to the ribosomal A-site Peptidyl transferase joins the tRNA at the P-site to the aa linked to the tRNA at the A-site with a peptide bond The new peptidyl-tRNA is translocated from the A-site to the P-site Phase 2: Elongation Protein Synthesis

Elongation With the formation of the 70S initiation complex, the elongation cycle can begin. With the formation of the 70S initiation complex, the elongation cycle can begin. Elongation involves the three factors, EF-Tu, EF-Ts, EF-G, as well as GTP, charged tRNA and the 70S initiation complex. Elongation involves the three factors, EF-Tu, EF-Ts, EF-G, as well as GTP, charged tRNA and the 70S initiation complex. The protein elongation cycle in prokaryotes and eukaryotes is quite similar. The factors EF-Tu EF-Ts EF-G have direct eukaryotic equivalents called eEF1α eEF1βγ eEF2

Polypeptide tRNA with amino acid attached Ribosome tRNA Anticodon 3 5 mRNA Amino acids Codons Elongation - Amino acids are added one by one to the preceding amino acid -Elongation factors facilitate - codon recognition - peptide bond formation - translocation

Amino end mRNA 5 3 Growing polypeptide Next amino acid to be added to polypeptide chain tRNA Codons Schematic model with mRNA and tRNA E Ribosomes translate 5’ to 3’ on mRNA. Polypeptide chains grow amino end first, carboxyl end last. The ribosome moves down one codon at a time. The now-empty tRNA at the P site is displaced off the ribosome through the E site. The process is then repeated: The tRNA at the P site holds the peptide chain, and a new tRNA binds to the A site. the peptide chain is transferred onto the amino acid attached to the A site tRNA. The ribosome moves down one codon, displacing the empty P site tRNA and moving the tRNA with the peptide chain from the A site to the P site.

Ribosome ready for next aminoacyl tRNA mRNA 5 Amino end of polypeptide E P site A site GDP E PA GTP GDP E PA E PA 1. Recognition 2. Peptide bond formation 3. Translocation

Copyright © 2006 by Elsevier, Inc. Release factor binds to the stop codon. Completed polypeptide is released. The ribosome dissociates into its 2 subunits. Phase 3: Termination Protein Synthesis

3 The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. The two ribosomal subunits and the other components of the assembly dissociate. Release factor Stop codon (UAG, UAA, or UGA) Free polypeptide When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA. Termination

Occurs when the A site of ribosomes reaches a stop codon in the mRNA Three different codons are used as “stop codons”. They code for no amino acid, and all protein-coding regions end in a stop codon. When the ribosome reaches a stop codon, there is no tRNA that binds to it. Instead, proteins called “release factors” bind and cause the ribosome, the mRNA, and the new polypeptide to dissociate. The new polypeptide is completed. Release factor causes addition of water molecule instead of amino acid Termination Note that the mRNA continues on past the stop codon. The remaining portion is not translated: it is the 3’ untranslated region (3’ UTR).

Termination in eukaryote Eukaryotes use only one release factors eRF, which requires GTP, recognizes all three termination codons. Termination codon is one of three (UAG, UAA, UGA) that causes protein synthesis to terminate.

prokaryoticeukaryoticfunction Initiation factor IF1 IF3 IF2 eIF3 eIF4c eIF6 eIF4B eIF4F eIF2B eIF2 eIF5 Bind to ribosome submits Bind to mRNA Initiator tRNA delivery Displacement of other factors Elongation factor EF-Tu EF-Ts EF-g eEF1α eEF1βγ eEF2 Aminoacyl tRNA delivery Recycling of EF-Tu or eEF1α Translocation Termination factors RF1, RF2, RF3eRFPolypeptides Chain release

Quiz Monday, May 8 th 3:45-5:00 40 questions: 15 genetics 25 biochemistry